Engineered nucleic acids-targeting nucleic acids

ABSTRACT

The present disclosure provides engineered polynucleotide sequences that form scaffolds and nucleoprotein complexes comprising such engineered polynucleotide sequences that form scaffolds and nucleic acid binding proteins. Nucleic acid sequences encoding the engineered polynucleotide sequences that form scaffolds, as well as expression cassettes, vectors and cells comprising such polynucleotide sequences, are described. A variety of methods for making and using the engineered polynucleotide sequences that form scaffolds are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/263,232, filed 4 Dec. 2015, now pending, whichapplication is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

SEQUENCE LISTING

The present application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on 2 Dec. 2016 isnamed CBI020-10_ST25.txt and is 156 KB in size.

TECHNICAL FIELD

The present disclosure relates generally to engineered polynucleotidesequences that form scaffolds and nucleoprotein complexes comprisingsuch scaffolds and nucleic acid binding proteins. Nucleic acid sequencesencoding the scaffold polynucleotide components, as well as expressioncassettes, vectors, and cells comprising the polynucleotide componentsare described. The disclosure also relates to methods for making andusing the engineered nucleic acid sequences that form scaffolds and thenucleoprotein complexes of the present invention.

BACKGROUND

Clustered regularly interspaced short palindromic repeats (CRISPR) andCRISPR-associated proteins (Cas) constitute the CRISPR-Cas system. TheCRISPR-Cas system provides adaptive immunity against foreign DNA inbacteria (see, e.g., Barrangou, R., et al., Science 315:1709-1712(2007); Makarova, K. S., et al., Nature Reviews Microbiology 9:467-477(2011); Garneau, J. E., et al., Nature 468:67-71 (2010); Sapranauskas,R., et al., Nucleic Acids Research 39:9275-9282 (2011)).

CRISPR-Cas systems have recently been reclassified into two classes,comprising five types and sixteen subtypes (see Makarova, K., et al.,Nature Reviews Microbiology 13:1-15 (2015)). This classification isbased upon identifying all Cas genes in a CRISPR-Cas locus anddetermining the signature genes in each CRISPR-Cas locus, ultimatelyplacing the CRISPR-Cas systems in either Class 1 or Class 2 based uponthe genes encoding the effector module, i.e., the proteins involved inthe interference stage. Recently a sixth CRISPR-Cas system (Type VI) hasbeen identified (see Abudayyeh 0., et al., Science 353(6299):aaf5573(2016)). Certain bacteria possess more than one type of CRISPR-Cassystem.

Class 1 systems have a multi-subunit crRNA-effector complex, whereasClass 2 systems have a single protein, such as Cas9, Cpf1, C2c1, C2c2,C2c3, or a crRNA-effector complex. Class 1 systems comprise Type I, TypeIII, and Type IV systems. Class 2 systems comprise Type II, Type V, andType VI systems.

Type II systems have cas1, cas2, and cas9 genes. The cas9 gene encodes amulti-domain protein that combines the functions of the crRNA-effectorcomplex with DNA target sequence cleavage. Type II systems are furtherdivided into three subtypes, subtypes II-A, II-B, and II-C. Subtype II-Acontains an additional gene, csn2. Examples of organisms with a subtypeII-A systems include, but are not limited to, Streptococcus pyogenes,Streptococcus thermophilus, and Staphylococcus aureus. Subtype II-Blacks the csn2 protein, but has the cas4 protein. An example of anorganism with a subtype II-B system is Legionella pneumophila. SubtypeII-C is the most common Type II system found in bacteria and has onlythree proteins, Cas1, Cas2, and Cas9. An example of an organism with asubtype II-C system is Neisseria lactamica.

Type V systems have a cpf1 gene and cas1 and cas2 genes (see Zetsche,B., et al., Cell 163:1-13 (2015)). The cpf1 gene encodes a protein,Cpf1, that has a RuvC-like nuclease domain that is homologous to therespective domain of Cas9, but lacks the HNH nuclease domain that ispresent in Cas9 proteins. Type V systems have been identified in severalbacteria including, but not limited to, Parcubacteria bacterium,Lachnospiraceae bacterium, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium, Acidaminococcus spp., Porphyromonasmacacae, Porphyromonas crevioricanis, Prevotella disiens, Moraxellabovoculi, Smithella spp., Leptospira inadai, Franciscella tularensis,Franciscella novicida, Candidatus methanoplasma termitum, andEubacterium eligens. Recently it has been demonstrated that Cpf1 alsohas RNase activity and is responsible for pre-crRNA processing (seeFonfara, I., et al., Nature 532(7600):517-521 (2016)).

In Class 2 systems, the crRNA is associated with a single protein andachieves interference by combining nuclease activity with RNA-bindingdomains and base-pair formation between the crRNA and a nucleic acidtarget sequence.

In Type II systems, nucleic acid target sequence binding involves Cas9and the crRNA, as does nucleic acid target sequence cleavage. In Type IIsystems, the RuvC-like nuclease (RNase H fold) domain and the HNH(McrA-like) nuclease domain of Cas9 each cleave one of the strands ofthe double-stranded nucleic acid target sequence. The Cas9 cleavageactivity of Type II systems also requires hybridization of crRNA to atracrRNA to form a duplex that facilitates the crRNA and nucleic acidtarget sequence binding by the Cas9 protein.

In Type V systems, nucleic acid target sequence binding involves Cpf1and the crRNA, as does nucleic acid target sequence cleavage. In Type Vsystems, the RuvC-like nuclease domain of Cpf1 cleaves one strand of thedouble-stranded nucleic acid target sequence, and a putative nucleasedomain cleaves the other strand of the double-stranded nucleic acidtarget sequence in a staggered configuration, producing 5′ overhangs,which is in contrast to the blunt ends generated by Cas9 cleavage.

The Cpf1 cleavage activity of Type V systems does not requirehybridization of crRNA to tracrRNA to form a duplex, rather the crRNA ofType V systems uses a single crRNA that has a stem-loop structureforming an internal duplex. Cpf1 binds the crRNA in a sequence andstructure specific manner that recognizes the stem loop and sequencesadjacent to the stem loop, most notably the nucleotides 5′ of the spacersequences that hybridizes to the nucleic acid target sequence. Thisstem-loop structure is typically in the range of 15 to 19 nucleotides inlength. Substitutions that disrupt this stem-loop duplex abolishcleavage activity, whereas other substitutions that do not disrupt thestem-loop duplex do not abolish cleavage activity. Nucleotides 5′ of thestem loop adopt a pseudo-knot structure further stabilizing thestem-loop structure with non-canonical Watson-Crick base pairing,triplex interaction, and reverse Hoogsteen base pairing (see Yamano, T.,et al., Cell 165(4):949-962 (2016)). In Type V systems, the crRNA formsa stem-loop structure at the 5′ end, and the sequence at the 3′ end iscomplementary to a sequence in a nucleic acid target sequence.

Other proteins associated with Type V crRNA and nucleic acid targetsequence binding and cleavage include Class 2 candidate 1 (C2c1) andClass 2 candidate 3 (C2c3). C2c1 and C2c3 proteins are similar in lengthto Cas9 and Cpf1 proteins, ranging from approximately 1,100 amino acidsto approximately 1,500 amino acids. C2c1 and C2c3 proteins also containRuvC-like nuclease domains and have an architecture similar to Cpf1.C2c1 proteins are similar to Cas9 proteins in requiring a crRNA and atracrRNA for nucleic acid target sequence binding and cleavage but havean optimal cleavage temperature of 50° C. C2c1 proteins target anAT-rich protospacer adjacent motif (PAM), similar to the PAM of Cpf1,which is 5′ of the nucleic acid target sequence (see, e.g., Shmakov, S.,et al., Molecular Cell 60(3):385-397 (2015)).

Class 2 candidate 2 (C2c2) does not share sequence similarity with otherCRISPR effector proteins and was recently identified as a Type VI system(see Abudayyeh, O., et al., Science 353(6299):aaf5573 (2016)). C2c2proteins have two HEPN domains and demonstrate single-stranded RNAcleavage activity. C2c2 proteins are similar to Cpf1 proteins inrequiring a crRNA for nucleic acid target sequence binding and cleavage,although not requiring tracrRNA. Also, similar to Cpf1, the crRNA forC2c2 proteins forms a stable hairpin, or stem-loop structure, that aidsin association with the C2c2 protein. Type VI systems have a singlepolypeptide RNA endonuclease that utilizes a single crRNA to directsite-specific cleavage. Additionally, after hybridizing to the targetRNA complementary to the spacer, C2c2 becomes a promiscuous RNAendonuclease exhibiting non-specific endonuclease activity toward anysingle-stranded RNA in a sequence independent manner (see East-Seletsky,A., et al., Nature 538(7624):270-273 (2016)).

Regarding Class 2 Type II CRISPR-Cas systems, a large number of Cas9orthologs are known in the art as well as their associatedpolynucleotide components (tracrRNA and crRNA) (see, e.g., Fonfara, I.,et al., Nucleic Acids Research 42(4):2577-2590 (2014), including allSupplemental Data; Chylinski K., et al., Nucleic Acids Research42(10):6091-6105 (2014), including all Supplemental Data). In addition,Cas9-like synthetic proteins are known in the art (see U.S. PublishedPatent Application No. 2014-0315985, published 23 Oct. 2014).

Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 is an endonucleasethat can be programmed by the tracrRNA/crRNA to cleave, in asite-specific manner, a DNA target sequence using two distinctendonuclease domains (HNH and RuvC/RNase H-like domains) (see U.S.Published Patent Application No. 2014-0068797, published 6 Mar. 2014;see also Jinek, M., et al., Science 337:816-821 (2012)).

Typically, each wild-type CRISPR-Cas9 system includes a crRNA and atracrRNA. The crRNA has a region of complementarity to a potential DNAtarget sequence and a second region that forms base-pair hydrogen bondswith the tracrRNA to form a secondary structure, typically to form atleast one stem structure. The region of complementarity to the DNAtarget sequence is the spacer. The tracrRNA and a crRNA interact througha number of base-pair hydrogen bonds to form secondary RNA structures.Complex formation between tracrRNA/crRNA and Cas9 protein results inconformational change of the Cas9 protein that facilitates binding toDNA, endonuclease activities of the Cas9 protein, and crRNA-guidedsite-specific DNA cleavage by the endonuclease Cas9. For a Cas9protein/tracrRNA/crRNA complex to cleave a double-stranded DNA targetsequence, the DNA target sequence is adjacent to a cognate PAM. Byengineering a crRNA to have an appropriate spacer sequence, the complexcan be targeted to cleave at a locus of interest, e.g., a locus at whichsequence modification is desired.

A variety of Type II CRISPR-Cas system crRNA and tracrRNA sequences, aswell as predicted secondary structures are known in the art (see, e.g.,Ran, F.A., et al., Nature 520(7546):186-191 (2015), including allSupplemental Data, in particular Extended Data FIG. 1; Fonfara, I., etal., Nucleic Acids Research 42(4):2577-2590 (2014), including allSupplemental Data, in particular Supplemental Figure S11). PredictedtracrRNA secondary structures were based on the Constraint GenerationRNA folding model (Zuker, M., Nucleic Acids Research 31:3406-3415(2003). RNA duplex secondary structures were predicted using RNAcofoldof the Vienna RNA package (Bernhart, S. H., et al., Algorithms forMolecular Biology 1(1):3 (2006); Hofacker, I. L., et al., Journal ofMolecular Biology 319:1059-1066 (2002)) and RNAhybrid(bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The structurepredictions were visualized using VARNA (Darty, K., et al.,Bioinformatics 25:1974-1975 (2009)). Fonfara, I., et al., show that thecrRNA/tracrRNA complex for Campylobacter jejuni does not have the bulgeregion; however, the complex retains a stem structure located 3′ of thespacer that is followed in the 3′ direction with another stem structure.

The spacer of Class 2 CRISPR-Cas systems can hybridize to a nucleic acidtarget sequence that is located 5′ or 3′ of a PAM, depending upon theCas protein to be used. A PAM can vary depending upon the Caspolypeptide to be used. For example, if Cas9 from S. pyogenes is used,the PAM can be a sequence in the nucleic acid target sequence thatcomprises the sequence 5′-NRR-3′, wherein R can be either A or G, N isany nucleotide, and N is immediately 3′ of the nucleic acid targetsequence targeted by the nucleic acid target binding sequence. A Casprotein may be modified such that a PAM may be different compared with aPAM for an unmodified Cas protein. If, for example, Cas9 from S.pyogenes is used, the Cas9 protein may be modified such that the PAM nolonger comprises the sequence 5′-NRR-3′, but instead comprises thesequence 5′-NNR-3′, wherein R can be either A or G, N is any nucleotide,and N is immediately 3′ of the nucleic acid target sequence targeted bythe nucleic acid target sequence.

Other Cas proteins recognize other PAMs, and one of skill in the art isable to determine the PAM for any particular Cas protein. For example,Cpf1 has a thymine-rich PAM site that targets, for example, a TTTNsequence (see Fagerlund, R., et al., Genome Biology 16:251 (2015)).

The RNA-guided Cas9 endonuclease has been widely used for programmablegenome editing in a variety of organisms and model systems (see, e.g.,Jinek M., et al., Science 337:816-821 (2012); Jinek M., et al., eLife2:e00471. doi: 10.7554/eLife.00471 (2013); U.S. Published PatentApplication No. 2014-0068797, published 6 Mar. 2014).

Genome engineering includes altering the genome by deleting, inserting,mutating, or substituting specific nucleic acid sequences. Thealteration can be gene- or location-specific. Genome engineering can usesite-directed nucleases, such as Cas proteins and their cognatepolynucleotides, to cut DNA, thereby generating a site for alteration.In certain cases, the cleavage can introduce a double-strand break (DSB)in the DNA target sequence. DSBs can be repaired, e.g., bynon-homologous end joining (NHEJ), microhomology-mediated end joining(MMEJ), or homology-directed repair (HDR). HDR relies on the presence ofa template for repair. In some examples of genome engineering, a donorpolynucleotide or portion thereof can be inserted into the break.

SUMMARY OF THE INVENTION

The present invention relates generally to a nucleic acid polynucleotidecomposition comprising a polynucleotide complex forming a scaffold thatis capable of binding a nucleic acid binding protein. Typically, a NASCpolynucleotide composition is a complex of two or more engineerednucleic acid sequences forming a scaffold comprising: a repeat element1, a repeat element 2 a nucleic acid binding protein binding element 1,a nucleic acid binding protein binding element 2, a spacer element 1(e.g., comprising a nucleic acid target binding sequence, and a spacerelement 2 (e.g., comprising a nucleic acid target binding sequence 2).The NASC polynucleotide composition is capable of associating with anucleic acid binding protein.

In an aspect, the present invention relates to a composition of two ormore engineered nucleic acid sequences forming a scaffold (“NASC”)comprising a first engineered nucleic acid “NASC-PC1” and a secondengineered nucleic acid component (“NASC-PC2”). The NASC-P1 comprises,in a 5′ to 3′ direction, a spacer element 1 comprising a nucleic acidtarget binding sequence 1, a repeat element 1 comprising a repeatnucleic acid sequence 1, and a nucleic acid binding protein bindingelement 1, wherein the spacer element 1 is covalently connected with therepeat element 1, and the repeat element 1 is covalently connected withthe nucleic acid binding protein binding element 1 comprising a nucleicacid binding protein binding sequence 1. The second engineered nucleicacid component (“NASC-PC2”) comprises, in a 5′ to 3′ direction, a spacerelement 2 comprising a nucleic acid target binding sequence 2, a repeatelement 2 comprising a repeat nucleic acid sequence 2, and a nucleicacid binding protein binding element 2 comprising a nucleic acid bindingprotein binding sequence 2, wherein the spacer element 2 is covalentlyconnected with the repeat element 2, and the repeat element 2 iscovalently connected with the nucleic acid binding protein bindingelement 2. In some embodiments of the present invention, the nucleicacid binding protein binding sequence 1 comprises a double-strandednucleic acid binding protein binding sequence 1, and the nucleic acidbinding protein binding sequence 2 comprises a double-stranded nucleicacid binding protein binding sequence 2. The repeat nucleic acidsequence 1 and the repeat nucleic acid sequence 2 are connected throughhydrogen-bonded base pairs and the connection forms the NASCcomposition. The NASC composition is capable of binding a first nucleicacid binding protein (e.g., a first double-stranded nucleic acid bindingprotein) and a second nucleic acid binding protein (e.g., a seconddouble-stranded nucleic acid binding protein).

Embodiments of the NASC composition include, but are not limited to, thefirst double-stranded nucleic acid binding protein being a Class 2CRISPR protein and the second double-stranded nucleic acid bindingprotein being a Class 2 CRISPR protein. In preferred embodiments, afirst double-stranded nucleic acid binding protein is a Class 2 Type IICRISPR-Cas9 protein, and a second double-stranded nucleic acid bindingprotein is a Class 2 Type II CRISPR-Cas9 protein. Other embodimentsinclude wherein the first double-stranded nucleic acid binding proteinis a Class 2 Type V CRISPR-Cpf1 protein, and wherein the seconddouble-stranded nucleic acid binding protein is a Class 2 Type VCRISPR-Cpf1 protein. In further embodiments, a first double-strandednucleic acid binding protein is a Class 2 Type II CRISPR-Cas9 protein,and a second double-stranded nucleic acid binding protein is a Class 2Type V CRISPR-Cpf1 protein.

In some embodiments, the spacer element 1 and spacer element 2 compriseadditional nucleic acid sequences. For example, the spacer element 1 canfurther comprise a linker element nucleic acid sequence 3′ of thenucleic acid target binding sequence 1 and 5′ of the repeat element 1.The spacer element 2 can further comprise a linker element nucleic acidsequence 3′ of the nucleic acid target binding sequence 1 and 5′ of therepeat element 1.

In further embodiments, the repeat element 1 and the repeat element 2comprise additional sequences as follows. The repeat element 1 furthercomprises, in a 5′ to 3′ direction, a repeat nucleic acid sequence 1b, alinker element nucleic acid sequence 1, and a repeat nucleic acidsequence 1a. The repeat element 2 further comprises, in a 5′ to 3′direction, a repeat nucleic acid sequence 1aC, a linker element nucleicacid sequence 2, and a repeat nucleic acid sequence 1bC. The repeatnucleic acid sequence 1b and the repeat nucleic acid sequence 1bC areconnected through hydrogen-bonded base pairs, and the repeat nucleicacid sequence 1a and the repeat nucleic acid sequence 1aC are connectedthrough hydrogen-bonded base pairs.

In additional embodiments, the repeat nucleic acid sequence 1b and therepeat sequence 1a comprise additional components as follows. The repeatnucleic acid sequence 1b can further comprise, in a 5′ to 3′ direction,a repeat nucleic acid sequence 1b2, a bulge nucleic acid sequence 1b1,and a repeat nucleic acid sequence 1b1. The repeat nucleic acid sequence1a can further comprise, in a 5′ to 3′ direction, a repeat nucleic acidsequence 1a2, a bulge nucleic acid sequence 1a1, and a repeat nucleicacid sequence 1a1. The repeat nucleic acid sequence 1aC can furthercomprise, in a 5′ to 3′ direction, a repeat nucleic acid sequence 1a1C,a bulge nucleic acid sequence 2a2, and a repeat nucleic acid sequence1a2C. The repeat nucleic acid sequence 1bC can further comprise, in a 5′to 3′ direction, a repeat nucleic acid sequence 1b1C, a bulge nucleicacid sequence 2b2, and a repeat nucleic acid sequence 1b2C. The repeatnucleic acid sequence 1a1 and the repeat nucleic acid sequence 1a1C areconnected through hydrogen-bonded base pairs, the repeat nucleic acidsequence 1a2 and the repeat nucleic acid sequence 1a2C are connectedthrough hydrogen-bonded base pairs, the repeat nucleic acid sequence 1b1and the repeat nucleic acid sequence 1b1C are connected throughhydrogen-bonded base pairs, and the repeat nucleic acid sequence 1b2 andthe repeat nucleic acid sequence 1b2C are connected throughhydrogen-bonded base pairs.

In additional embodiments, the linker element nucleic acid sequence 1-2and the linker element nucleotide sequence 2-2 comprise added nucleicacid sequences. The linker element 1-1 can comprise further comprise, ina 5′ to 3′ direction, a linker element nucleic acid sequence 1-2-2, arepeat nucleic acid sequence 1-2a, and a linker element nucleic acidsequence 1-2-1. The linker element nucleic acid sequence 2-2 can furthercomprise, in a 5′ to 3′ direction, a linker element nucleic acidsequence 2-2-1, a repeat nucleic acid sequence 1-2aC, and a linkerelement nucleic acid sequence 2-2-2. The repeat nucleic acid sequence1-2a and the repeat nucleic acid sequence 1-2aC are connected throughhydrogen-bonded base pairs and form a double-stranded nucleic acidregion 1-2. In some embodiments, the double-stranded nucleic acid region1-2 further comprises an effector protein binding site 1. The repeatnucleic acid sequence 1-2a further comprises an effector protein bindingsite nucleic acid sequence 1-2a. The repeat nucleic acid sequence 2further comprises an effector protein binding site nucleic acid sequence1-2aC. The effector binding site is formed by hydrogen base pair bondingbetween the effector protein binding site nucleic acid sequence 1-2a andthe effector protein binding site nucleic acid sequence 1-2aC. The Csy4protein binding site is an example of an effector protein binding site.A Csy4 protein or an enzymatically inactive Csy4 protein are capable ofbinding the effector binding site.

In further embodiments, the repeat nucleic acid sequence 1 furthercomprises an affinity tag 1 and the repeat nucleic acid sequence 2further comprises an affinity tag 2, and the affinity tag 1 is connectedwith affinity tag 2.

The NASC compositions can comprise, for example, RNA, DNA, or RNA andDNA. In some embodiments, NASC-PC1, NASC-PC2, or NASC-PC1 and NASC-PC2comprises RNA, DNA, or RNA and DNA.

In another aspect, the present invention includes a nucleic acid/proteincomposition comprising a NASC composition and one or more nucleic acidbinding proteins. In one embodiment, the nucleic acid proteins can be afirst Cas9 protein and a second Cas9 protein. For example, the firstCas9 protein is the same as the second Cas9 protein, and the first Cas9protein and the second Cas9 protein are selected from the groupconsisting of a S. pyogenes Cas9 protein, a S. thermophilus Cas9protein, a S. aureus Cas9 protein, and a C. jejuni Cas9 protein. Inother embodiments, the first Cas9 protein is different from the secondCas9 protein, and the first Cas9 protein and the second Cas9 protein areselected from the group consisting of a S. pyogenes Cas9 protein, a S.thermophilus Cas9 protein, a S. aureus Cas9 protein, and a C. jejuniCas9 protein. Additionally, the first Cas9 protein and the second Cas9protein can be selected from the group consisting of Cas9 protein/Cas9protein, Cas9 protein/dCas9 protein, dCas9 protein/Cas9 protein, anddCas9 protein/dCas9 protein, respectively.

In a further aspect, the present invention relates to kits comprisingone or more components of a NASC composition. In some embodiments, theNASC composition comprises a NASC-PC1 and a NASC-PC2, or one or morenucleic acid sequences encoding the NASC-PC1 and the NASC-PC2, and abuffer. Kits can further comprise one or more Cas9 proteins or one ormore nucleic acid sequences encoding the one or more Cas9 proteins. Infurther embodiments, a kit can comprise nucleoprotein complexescomprising a NASC composition and one or more Cas9 proteins.

In an additional aspect, the present invention relates to an expressionvector comprising one or more nucleic acid sequences encoding one ormore components of a NASC composition.

In yet another aspect, the present invention relates to a recombinantcell comprising one or more nucleic acid sequences encoding one or morecomponents of a NASC composition.

Further aspects of the present invention include methods of using NASCcomposition, as described herein. One method is a method of binding DNA.The method comprises contacting a first DNA target sequence in a DNApolynucleotide and a second DNA target sequence in the DNApolynucleotide with a nucleic acid/protein composition comprising NASCcomposition and a nucleic acid binding protein (e.g., a Cas9 protein,and/or a Cpf1 protein), thereby facilitating binding of the nucleicacid/protein composition to the first DNA target sequence in the DNA andthe second DNA target sequence in the DNA. A NASC-PC1 spacer element ofthe NASC composition can be complementary to the first DNA targetsequence, and the NASC-PC2 spacer of the NASC composition can becomplementary to the second DNA target sequence.

Another method of the present invention is a method of cutting DNA. Themethod comprises contacting a first DNA target sequence in the DNApolynucleotide and a second DNA target sequence in the DNApolynucleotide with a nucleic acid/protein composition comprising a NASCcomposition and a nucleic acid binding protein (e.g., a Cas9 protein,and/or a Cpf1 protein), thereby facilitating binding of the nucleicacid/protein composition to the first DNA target sequence and the secondDNA target sequence. Binding results in cutting of the first DNA targetsequence and the second DNA target sequence. A NASC-PC1 spacer elementof the NASC composition can be complementary to the first DNA targetsequence, and the NASC-PC2 spacer of the NASC composition can becomplementary to the second DNA target sequence.

These aspects and other embodiments of the present invention using theNASC compositions and nucleoprotein particles comprising the NASCcompositions of the present invention will be readily apparent to thoseof ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

The figures are not proportionally rendered, nor are the figures toscale. The locations of indicators are approximate.

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D present examples of dual-guideClass 2 Type II CRISPR-associated guide RNAs.

FIG. 2A, FIG. 2B, and FIG. 2C present examples of single-guide Class 2Type II CRISPR-associated guide RNAs.

FIG. 3A and FIG. 3B present examples of a Class 2 Type V crRNA guideRNAs.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H,FIG. 4I, FIG. 4J, FIG. 4K, FIG. 4L, FIG. 4M, FIG. 4N, and FIG. 4O (thislattermost figure is FIG. 4 “O” and not FIG. 4 “zero”) illustrateexamples of generic arrangements of engineered nucleic acid scaffoldpolynucleotide compositions of the present invention. The illustratedsequences are not rendered in a 5′ to 3′ or 3′ to 5′ orientation and donot have polarity.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H,and FIG. 5I illustrate examples and elements of engineered nucleic acidscaffold polynucleotide compositions of the present invention.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H,FIG. 6I, FIG. 6J, FIG. 6K, FIG. 6L, and FIG. 6M illustrate examples andelements of engineered nucleic acid scaffold polynucleotide compositionsof the present invention.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H,and FIG. 7I illustrate examples and elements of engineered concatenatednucleic acid scaffold polynucleotide compositions of the presentinvention.

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H,FIG. 8I, FIG. 8J, FIG. 8K, FIG. 8L, FIG. 8M, and FIG. 8N illustrateexamples and elements of engineered concatenated split-nexus nucleicacid scaffold polynucleotide compositions of the present invention.

FIG. 9A and FIG. 9B illustrate examples and elements of engineerednucleic acid scaffold polynucleotide compositions of the presentinvention.

FIG. 10 illustrates an example and elements of an engineered nucleicacid scaffold polynucleotide composition of the present invention.

FIG. 11 illustrates a nucleoprotein complex comprising an engineerednucleic acid scaffold polynucleotide composition of the presentinvention, formation of a nucleoprotein complex, and the nucleoproteincomplex binding two nucleic acid target sequences.

FIG. 12 illustrates a nucleoprotein complex comprising an engineerednucleic acid scaffold polynucleotide composition of the presentinvention binding to a first nucleic acid target sequence and binding toa second nucleic acid target sequence that is cut by a nuclease of thecomplex.

FIG. 13 illustrates a nucleoprotein complex comprising an engineerednucleic acid scaffold polynucleotide composition of the presentinvention binding to three nucleic acid target sequences in apolynucleotide.

FIG. 14 illustrates a nucleoprotein complex comprising an engineerednucleic acid scaffold polynucleotide composition of the presentinvention binding to a first nucleic acid target sequence in a firstpolynucleotide and binding to a second nucleic acid target sequence anda third nucleic acid target sequence in a second polynucleotide, whereinthe second and third nucleic acid target sequences are cut by a nucleaseof the complex.

FIG. 15 illustrates a nucleoprotein complex comprising an engineerednucleic acid scaffold polynucleotide composition of the presentinvention binding to a first nucleic acid target sequence in a firstpolynucleotide, binding to a second nucleic acid target sequence in asecond polynucleotide, and binding a third nucleic acid target sequencein a third polynucleotide, wherein the second and third nucleic acidtarget sequences are cut by a nuclease of the complex.

FIG. 16A, FIG. 16B, and FIG. 16C illustrate an engineered nucleic acidscaffold polynucleotide composition of the present invention forming anucleoprotein complex with two different proteins and binding to a firstnucleic acid target sequence in a first polynucleotide and a secondnucleic acid sequence in a second polynucleotide. Three combinations ofbinding and cleaving outcomes are illustrated.

INCORPORATION BY REFERENCE

All patents, publications, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpatent, publication, or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a polynucleotide” includes one or more polynucleotides, and referenceto “a vector” includes one or more vectors.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other methods andmaterials similar, or equivalent, to those described herein can beuseful in the present invention, preferred materials and methods aredescribed herein.

In view of the teachings of the present specification, one of ordinaryskill in the art can employ conventional techniques of immunology,biochemistry, chemistry, molecular biology, microbiology, cell biology,genomics, and recombinant polynucleotides, as taught, for example, bythe following standard texts: Antibodies: A Laboratory Manual, Secondedition, E. A. Greenfield, Cold Spring Harbor Laboratory Press, ISBN978-1-936113-81-1 (2014); Culture of Animal Cells: A Manual of BasicTechnique and Specialized Applications, 6th Edition, R. I. Freshney,Wiley-Blackwell, ISBN 978-0-470-52812-9 (2010); Transgenic AnimalTechnology, Third Edition: A Laboratory Handbook, C. A. Pinkert,Elsevier, ISBN 978-0124104907 (2014); The Laboratory Mouse, SecondEdition, H. Hedrich, Academic Press, ISBN 978-0123820082 (2012);Manipulating the Mouse Embryo: A Laboratory Manual, R. Behringer, etal., Cold Spring Harbor Laboratory Press, ISBN 978-1936113019 (2013);PCR 2: A Practical Approach, M. J. McPherson, et al., IRL Press, ISBN978-0199634248 (1995); Methods in Molecular Biology (Series), J. M.Walker, ISSN 1064-3745, Humana Press; RNA: A Laboratory Manual, D. C.Rio, et al., Cold Spring Harbor Laboratory Press, ISBN 978-0879698911(2010); Methods in Enzymology (Series), Academic Press; MolecularCloning: A Laboratory Manual (Fourth Edition), M. R. Green, et al., ColdSpring Harbor Laboratory Press, ISBN 978-1605500560 (2012); BioconjugateTechniques, Third Edition, G. T. Hermanson, Academic Press, ISBN978-0123822390 (2013); Methods in Plant Biochemistry and MolecularBiology, W. V. Dashek, CRC Press, ISBN 978-0849394805 (1997); Plant CellCulture Protocols (Methods in Molecular Biology), V. M. Loyola-Vargas,et al., Humana Press, ISBN 978-1617798177 (2012); Plant TransformationTechnologies, C. N. Stewart, et al., Wiley-Blackwell, ISBN978-0813821955 (2011); Recombinant Proteins from Plants (Methods inBiotechnology), C. Cunningham, et al., Humana Press, ISBN 978-1617370212(2010); Plant Genomics: Methods and Protocols (Methods in MolecularBiology), D. J. Somers, et al., Humana Press, ISBN 978-1588299970(2009); Plant Biotechnology: Methods in Tissue Culture and GeneTransfer, R. Keshavachandran, et al., Orient Blackswan, ISBN978-8173716164 (2008).

Clustered regularly interspaced short palindromic repeats (CRISPR) andrelated CRISPR-associated proteins (Cas proteins) constitute CRISPR-Cassystems (see, e.g., Barrangou, R., et al., Science 315:1709-1712(2007)).

As used herein, “Cas protein” and “CRISPR-Cas protein” refer to Casproteins including, but not limited to, Class 1 Type I Cas proteins,Class 1 Type III Cas proteins, Class 1 Type IV Cas proteins, Class 2Type II Cas proteins, Class 2 Type V Cas proteins, and Class 2 Type VICas proteins. Class 2 Cas proteins include Cas9 proteins, Cas9-likeproteins encoded by Cas9 orthologs, Cas9-like synthetic proteins, Cpf1proteins, proteins encoded by Cpf1 orthologs, Cpf1-like syntheticproteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, and variants andmodifications thereof. In some embodiments, Cas proteins are Class 2 Casproteins, for example one or more Class 2 Type II Cas proteins, such asCas9, one or more Class 2 Type V Cas proteins, such as Cpf1, or one ormore Class 2 Type VI Cas proteins, such as C2c2. In preferredembodiments, Cas proteins are one or more Class 2 Type II Cas proteins,such as Cas9, and one or more Class 2 Type V Cas proteins, such as Cpf1.Typically, for use in aspects of the present invention, a Cas protein iscapable of interacting with one or more cognate polynucleotides (mosttypically, RNA) to form a nucleoprotein complex (most typically, aribonucleoprotein complex).

“Cas9 protein,” as used herein, refers to a Cas9 wild-type proteinderived from Class 2 Type II CRISPR-Cas9 systems, modifications of Cas9proteins, variants of Cas9 proteins, Cas9 orthologs, and combinationsthereof. Cas9 proteins include, but not limited to, Cas9 fromStreptococcus pyogenes (UniProtKB-Q99ZW2 (CAS9_STRP1)), Streptococcusthermophilus (UniProtKB-G3ECR1 (CAS9_STRTR)), and Staphylococcus aureus(UniProtKB-J7RUA5 (CAS9_STAAU)). Cas9 homologs can be identified usingsequence similarity search methods known to one skilled in the art.“dCas9,” as used herein, refers to variants of Cas9 protein that arenuclease-deactivated Cas9 proteins, also termed “catalytically inactiveCas9 protein,” “enzymatically inactive Cas9,” “catalytically dead Cas9”or “dead Cas9.” Such molecules lack all or a portion of endonucleaseactivity and can therefore be used to regulate genes in an RNA-guidedmanner (see Jinek M., et al., Science 337:816-821 (2012)). This isaccomplished by introducing mutations to catalytic residues, such asD10A in the RuvC-1 domain and H840A in the HNH domain (numbered relativeto S. pyogenes Cas9 protein), that inactivate Cas9 nuclease function. Itis understood that mutation of other catalytic residues to reduceactivity of either or both of the nuclease domains can also be carriedout by one skilled in the art. The resultant dCas9 is unable to cleavedouble-stranded DNA but retains the ability to complex with a guidenucleic acid and bind a DNA target sequence. The Cas9 double mutant withchanges at amino acid positions D10A and H840A inactivates both thenuclease and nickase activities. Targeting specificity is determined byCas9 protein binding to the PAM sequence, and by complementary basepairing of guide RNA (typically, a single-guide RNA) to the genomiclocus. Cas9 is the signature protein characteristic for Class 2 Type IICRISPR systems.

“Cpf1 protein,” as used herein, refers to a Cpf1 wild-type proteinderived from Class 2 Type V CRISPR-Cpf1 systems, modifications of Cpf1proteins, variants of Cpf1 proteins, Cpf1 orthologs, and combinationsthereof. “dCpf1,” as used herein, refers to variants of Cpf1 proteinthat are nuclease-deactivated Cpf1 proteins, also termed “catalyticallyinactive Cpf1 protein,” or “enzymatically inactive Cpf1.” Cpf1 proteinsinclude, but not limited to, Francisella novicida (UniProtKB-A0Q7Q2(CPF1_FRATN)), Lachnospiraceae bacterium (UniProtKB-A0A182DWE3(A0A182DWE3_9FIRM)), and Acidaminococcus sp. (UniProtKB-U2UMQ6(CPF1_ACISB)). Cpf1 is the signature protein characteristic for Class 2Type V CRISPR systems. Cpf1 homologs can be identified using sequencesimilarity search methods known to one skilled in the art.

“Nucleic-acid targeting nucleic acid” (NATNA), as used herein, refers toone or more polynucleotides that guide a protein, such as a Cas protein(e.g., a Cas9 protein or a Cpf1 protein), to preferentially bind anucleic acid target sequence in a polynucleotide (relative to apolynucleotide that does not comprise the nucleic acid target sequence).NATNAs can comprise ribonucleotide bases (e.g., RNA),deoxyribonucleotide bases (e.g., DNA), combinations of ribonucleotidebases and deoxyribonucleotide bases (e.g., RNA/DNA), nucleotides,nucleotide analogs, modified nucleotides, and the like, as well assynthetic, naturally occurring, and non-naturally occurring modifiedbackbone residues or linkages, for example, as described herein.Examples of nucleic-acid targeting nucleic acids include, but are notlimited to, Cas9-crRNA/tracrRNA molecules (see, e.g., FIG. 1A, FIG. 1B,FIG. 1C, and FIG. 1D), Cas9-sgRNA (see, e.g., FIG. 2A and FIG. 2B), andCpf1-crRNA (see, e.g., FIG. 3A and FIG. 3B).

As used herein, “dual-guide RNA” and “Cas9-dual-guide RNA” typicallyrefer to a two-component RNA system for a polynucleotide componentcapable of associating with a cognate Cas9 protein. FIG. 1A and FIG. 1Bpresent illustrative examples of Class 2 Type II CRISPR-Cas9-associateddual-guide RNAs. FIG. 1A illustrates a Type II CRISPR-Cas9 systemtwo-component RNA comprising a Cas9-crRNA (FIG. 1A, 101) and aCas9-tracrRNA (FIG. 1A, 102). FIG. 1B illustrates the formation ofbase-pair hydrogen bonds between the Cas9-crRNA and the Cas9-tracrRNA toform secondary structure (see, e.g., U.S. Published Patent ApplicationNo. 2014-0068797, published 6 Mar. 2014; see also Jinek M., et al.,Science 337:816-21 (2012)). FIG. 1B presents an overview of andnomenclature for secondary structural elements of the Cas9-crRNA andCas9-tracrRNA of the S. pyogenes Cas9 including the following: a spacerelement (FIG. 1B, 103) comprising a spacer sequence (also referred toherein as a nucleic acid target binding sequence); a first stem element(FIG. 1B, 104, 105, 106) comprising a lower stem element (FIG. 1B, 104),a bulge element comprising unpaired nucleotides (FIG. 1B, 105), and anupper stem element (FIG. 1B, 106); a nexus element (FIG. 1B, 107)comprising a second stem element; a first 3′ hairpin element (FIG. 1B,108) comprising a third stem element; and a second 3′ hairpin element(FIG. 1B, 109) comprising a fourth stem element. In some Class 2 Type IICRISPR-Cas9 systems, the first stem element does not have a bulgeelement (e.g., C. jejuni). FIG. 1C illustrates a Type II CRISPR-Cas9two-RNA component system comprising a Cas9-crRNA (FIG. 1C, 101) and aCas9-tracrRNA (FIG. 1C, 102). FIG. 1D illustrates the formation ofbase-pair hydrogen bonds between the Cas9-crRNA and the Cas9-tracrRNA toform a secondary structure. FIG. 1D presents an overview of andnomenclature for the following: a spacer element (FIG. 1D, 103); a firststem element (FIG. 1D, 110); a nexus element (FIG. 1D, 107) comprising asecond stem element; a first 3′ hairpin element (FIG. 1D, 108)comprising a third stem element; and a second 3′ hairpin element (FIG.1D, 109) comprising a fourth stem element. A Cas9-dual-guide RNA iscapable of forming a nucleoprotein complex with a cognate Cas9 protein,wherein the complex is capable of targeting a nucleic acid targetsequence complementary to the spacer sequence. Modifications ofCas9-dual-guides are known in the art, including, deletion of one ormore 3′ hairpin elements (FIG. 1B, 108, 109; FIG. 1D, 108, 109),modifications of the first stem element (FIG. 1B, 104, 105, 106; FIG. 1D110), and modifications of the upper stem, bulge, and lower stem (FIG.1B, 106, 105, 104, respectively) (see, e.g., U.S. Patent Publication No.2014-0315985, published 23 Oct. 2014; U.S. Patent Publication No.2015-0376586, published 31 Dec. 2015). As used herein, a “dual-guideCas9 polynucleotide” refers to a two-component system having apolynucleotide with the same structural elements as a crRNA (FIG. 1A,101) and a polynucleotide with the same structural elements as atracrRNA (FIG. 1A 102). A dual-guide Cas9 polynucleotide system iscapable of associating with a cognate Cas9 protein.

As used herein, “single-guide RNA” (sgRNA) and “Cas9-sgRNA” typicallyrefer to a one-component RNA system as further described herein, whereinthe system is capable of associating with a cognate Cas9 protein.

FIG. 2A, FIG. 2B and FIG. 2C show examples of Class 2 Type IICRISPR-Cas9-associated RNA. These figures illustrate Cas9 single-guideRNAs (Cas9-sgRNA) wherein the Cas9-crRNA is covalently joined to theCas9-tracrRNA, often through a tetraloop, and forms a RNA polynucleotidesecondary structure through base-pair hydrogen bonding (see, e.g., U.S.Published Patent Application No. 2014-0068797, published 6 Mar. 2014).FIG. 2A presents an overview of and nomenclature for secondarystructural elements of a Cas9-sgRNA for S. pyogenes including thefollowing: a spacer element (FIG. 2A, 201) comprising a spacer sequence(also referred to herein as a nucleic acid targeting nucleic acidsequence); a first stem-loop element (FIG. 2A, 202, 205, 203, 204)comprising a lower stem element (FIG. 2A, 202), a bulge elementcomprising unpaired nucleotides (FIG. 2A, 205), and an upper stemelement (FIG. 2A, 203), and a loop element (FIG. 2A, 204) comprisingunpaired nucleotides; a nexus element (FIG. 2A, 206) comprising a secondstem-loop element; a first 3′ hairpin element (FIG. 2A, 207) comprisinga third stem-loop element; and a second 3′ hairpin element comprising athird stem element (FIG. 2A, 208) comprising a fourth stem-loop element.(See, e.g., FIGS. 1 and 3 of Briner, A. E., et al., Molecular Cell56(2):333-339 (2014)).

FIG. 2B presents an overview of and nomenclature for secondarystructural elements of a Cas9-sgRNA for C. jejuni including thefollowing: a spacer element (FIG. 2B, 201); a first stem element (FIG.2B, 209) and a loop element (FIG. 2B, 204) comprising unpairednucleotides (i.e., the first stem-loop element comprises the first stemelement and the loop element); a nexus element (FIG. 2B, 206) comprisinga second stem-loop element; a first 3′ hairpin element (FIG. 2B, 207)comprising a third stem-loop element; and a second 3′ hairpin elementcomprising a third stem element (FIG. 2B, 208) comprising a fourthstem-loop element. A Cas9-sgRNA is capable of forming a nucleoproteincomplex with a cognate Cas9 protein, wherein the complex is capable oftargeting a nucleic acid sequence complementary to the spacer sequence.

Modifications of Cas9 single-guides are known in the art including, butnot limited to, deletion of one or more 3′ hairpin elements (FIG. 2,207, 208), modifications of the first stem element (FIG. 1B, 104, 105,106; FIG. 1D 110), and modifications of the upper stem, bulge, and lowerstem (FIG. 1B, 106, 105, 104, respectively) (see, e.g., U.S. PatentPublication No. 2014-0315985, published 23 Oct. 2014; U.S. PatentPublication No. 2015-0376586, published 31 Dec. 2015).

As used herein, a “Cas9 single-guide polynucleotide” refers to aone-component system having the same structural elements as a sgRNA(FIG. 2). A single-guide Cas9 polynucleotide system is capable ofassociating with a cognate Cas9 protein.

FIG. 2C presents a more detailed illustration of FIG. 2A. Table 1presents a series of numerical indicators used to illustrate regions ofnucleic acid sequences associated with a Class 2 Type II CRISPR-Cas9sgRNA. In Table 1, “:” is the equivalent of the term “comprising.”

TABLE 1 Numerical Indicators Used to Illustrate Regions of Nucleic AcidSequences in a sgRNA Indicator Corresponding Region Nucleic acid bindingprotein binding sequences 210 a 3′ terminus 210-211 a linker elementnucleic acid sequence: a 3′ terminal linker element nucleic acidsequence 3′ hairpin elements (compare FIG. 2A, 207, 208) 211-214 ahairpin nucleic acid sequence 1-2: a 3′ hairpin 1-2 element 211-212 a 3′hairpin 1-2 stem element nucleic acid sequence 2 212-213 a 3′ hairpin1-2 loop element nucleic acid sequence 213-214 a 3′ hairpin 1-2 stemelement nucleic acid sequence 1 214-215 a linker element nucleic acidsequence: a 3′ hairpin 1-2 linker element nucleic acid sequence 215-218a hairpin nucleic acid sequence 1-1: a 3′ hairpin-1-1 element 215-216 a3′ hairpin 1-1 stem element nucleic acid sequence 2 216-217 a 3′ hairpin1-1 loop element nucleic acid sequence 217-218 a 3′ hairpin 1-1 stemelement nucleic acid sequence 1 218-219 a linker element nucleic acidsequence: a 3′ hairpin 1-1 linker element nucleic acid sequence Nexuselement 219-220 a nexus element nucleic acid sequence 219-230 asplit-nexus stem element nucleic acid sequence 1-1 230-220 a split-nexusstem element nucleic acid sequence 1-2 Stem 1 linker element 220-221 aconnective nucleic acid sequence 1: a linker element nucleic acidsequence 1 221-228 first stem-loop element (see also FIG. 2A, 202, 203,204, 205; FIG. 2B, 204, 209) 221-224/ first stem element (see also, FIG.1A, 104, 105, 106; 225-228 FIG. 1D, 110; 2A, 202, 205, 203) 221-222/ alower stem element 1 227-228 222-223/ a bulge element 1 226-227 223-224/an upper stem element 1 225-226 221-224 a first stem element nucleicacid sequence 1-2 221-222 a lower stem element nucleic acid sequence 1-2222-223 a bulge element nucleic acid sequence 1-2 223-224 an upper stemelement nucleic acid sequence 1-2 224-225 a loop element nucleic acidsequence 1 224-225 an upper stem element nucleic acid sequence 1-1224-225 a bulge element nucleic acid sequence 1-1 224-225 a lower stemelement nucleic acid sequence 1-1 225-228 a first stem element nucleicacid sequence 1-1 228-229 a spacer element 1 comprising a nucleic acidsequence 1: a spacer nucleic acid sequence: a nucleic acid targetbinding sequence (e.g., a DNA target binding sequence) 229 a 5′ end

“Class 2 Type V guide crRNA” and “Cpf1-crRNA,” as used herein, typicallyrefer to a one-component RNA system for a polynucleotide componentcapable of associating with a cognate Cpf1 protein (see, e.g., Zetsche,B., et al., Cell 163:1-13 (2015)). FIG. 3A presents an example of a TypeV CRISPR-Cpf1-associated RNA (Cpf1-crRNA), as well as an overview of andnomenclature for secondary structural elements of a Cpf1-crRNA asfollows: a stem-loop element (FIG. 3A, 301) and a spacer element (FIG.3A, 302) comprising a nucleic acid target binding sequence. Thestem-loop element comprises, in a 5′ to 3′ direction, a Cpf1-stem RNAsequence 1C (FIG. 3A, 303), a loop element (FIG. 3A, 304), and acomplementary Cpf1-stem RNA sequence 1C (FIG. 3A, 305), wherein theCpf1-stem RNA sequence 1 and the complementary Cpf1-stem RNA sequence 1Cform a duplex. FIG. 3B presents a modification of the Cpf1-crRNA whereinthe loop element is removed from the stem-loop element of FIG. 3A. FIG.3B illustrates a stem element (FIG. 3B, 301) comprising, in a 5′ to 3′direction, a Cpf1-stem nucleic acid sequence 1 (FIG. 3B, 303); acomplementary Cpf1-stem nucleic acid sequence 1C (FIG. 3B, 305), whereinthe Cpf1-stem nucleic acid sequence 1 and the complementary Cpf1-stemnucleic acid sequence 1C form a duplex; and a spacer element (FIG. 3A,302) comprising a nucleic acid target binding sequence. A guide crRNA iscapable of forming a nucleoprotein complex with a cognate Cpf1 protein,wherein the complex is capable of targeting a nucleic acid targetsequence complementary to the nucleic acid target binding sequence.

As used herein, “a nucleic acid target binding sequence” and “spacernucleic acid sequence” refer to nucleic acid sequences capable ofhybridizing to a nucleic acid target sequence in a polynucleotide. A“spacer element” comprises a nucleic acid target binding sequence.

As used herein, “a nucleic acid scaffold,” “NASC,” “a NASCpolynucleotide composition,” “a NASC composition” and “a NASCpolynucleotide composition” all refer to a polynucleotide complexforming a scaffold. In preferred embodiments, the scaffold is capable ofbinding a nucleic acid binding protein. Typically, a NASC polynucleotidecomposition is a complex of two or more engineered nucleic acidsequences forming a scaffold comprising: (i) a repeat element 1 (e.g.,comprising a repeat nucleic acid sequence 1) and a repeat element 2(e.g., comprising a repeat nucleic acid sequence 2); (ii) a nucleic acidbinding protein binding element 1 (e.g., comprising a nucleic acidbinding protein binding sequence 1) and a nucleic acid binding proteinbinding element 2 (e.g., comprising a nucleic acid binding proteinbinding sequence 2); and (iii) a spacer element 1 (e.g., comprising anucleic acid target binding sequence 1) and a spacer element 2 (e.g.,comprising a nucleic acid target binding sequence 2). In a NASCpolynucleotide composition, the repeat element 1 is connected with therepeat element 2.

The NASC polynucleotide composition is capable of associating with anucleic acid binding protein. In some embodiments, the NASCpolynucleotide composition is capable of associating with two or morenucleic acid binding proteins (e.g., nucleic acid binding proteinshaving similar structural motifs and functional motifs) to form anucleoprotein complex. Examples of nucleic acid binding proteins arediscussed herein below.

In some embodiments of NASC polynucleotide compositions, each of a firstNASC polynucleotide component (e.g., a NASC-PC1 comprising a repeatelement 1, a nucleic acid binding protein binding element 1, and aspacer element 1) and a second NASC polynucleotide component (e.g.,NASC-PC2 comprising a repeat element 2, a nucleic acid binding proteinbinding element 2, and a spacer element 2) is capable of associatingwith the same kind of a nucleic acid binding protein (e.g., nucleic acidbinding proteins having similar structural motifs and functional motifs)to form a nucleoprotein complex.

In other embodiments of NASC polypeptide compositions, a nucleoproteincomplex is capable of being formed by a nucleic acid binding proteinbinding to a macromolecule comprising a nucleic acid target bindingsequence 1, a repeat nucleic acid sequence 1, a repeat nucleic acidsequence 2, and a nucleic acid target binding sequence 1.

A NASC polynucleotide composition/nucleic acid binding protein 1/nucleicacid binding protein 2 complex is capable of preferentially binding anucleic acid target sequence in a polynucleotide (relative to apolynucleotide that does not comprise the nucleic acid target sequence).

A NASC polynucleotide (NASC-PC) comprising multiple spacer elements isgenerically referred to herein as “a NASC-PC-MTS,” and specificallyreferred to with reference to the number of spacer elements “aNASC-PC-(number of spacer elements)TS” (e.g., for two spacer elements,the designation used is a NASC-PC-2TS).

The components of a NASC-PC comprising multiple polynucleotides arereferred to herein with reference to the number of polynucleotides “aNASC-PC-(number of polynucleotides)” (e.g., for two polynucleotides, thedesignation used is a NASC-PC1-1 and a NASC-PC1-2).

A NASC-PC polynucleotide component comprising concatenated elements isreferred to herein as “NASC-PC-CE.” In particular embodiments comprisingsplit-nexus polynucleotides, a NASC polynucleotide component comprisingconcatenated split-nexus elements is referred to herein as“NASC-PC-SCE.”

As used herein, “a nucleic acid brace sequence” is a nucleic acidsequence comprising at least two distinct nucleic acid target sequences:a nucleic acid target sequence 1 complementary to a nucleic acid targetbinding sequence 1 of a first NASC polynucleotide composition, and anucleic acid target sequence 2 complementary to a nucleic acid targetbinding sequence 2 of a second NASC polynucleotide composition. Anexample of a nucleic acid brace sequence is a DNA brace sequence.

As used herein, “a NASC-Cage Composition (NASC-CC)” comprises at least afirst NASC polynucleotide composition connected by nucleic acid bracesequences to a second NASC polynucleotide composition to form acage-like structure typically having an internal space for packagingmolecules.

As used herein, the term “cognate” typically refers to a Cas protein(e.g., Cas9 protein or a Cpf1 protein) and one or more Caspolynucleotides (e.g., Class 2 Type II CRISPR-Cas9-associated NATNA orClass 2 Type V CRISPR-Cpf1-associated NATNAs, respectively) that arecapable of forming a nucleoprotein complex capable of site-directedbinding to a nucleic acid target sequence complementary to the nucleicacid target binding sequence present in one of the one or more Caspolynucleotides.

The terms “wild-type,” “naturally occurring,” and “unmodified” are usedherein to mean the typical (or most common) form, appearance, phenotype,or strain existing in nature; for example, the typical form of cells,organisms, characteristics, polynucleotides, proteins, macromolecularcomplexes, genes, RNAs, DNAs, or genomes as they occur in, and can beisolated from, a source in nature. The wild-type form, appearance,phenotype, or strain serve as the original parent before an intentionalmodification. Thus, mutant, variant, engineered, recombinant, andmodified forms are not wild-type forms.

As used herein, the terms “engineered,” “genetically engineered,”“recombinant,” “modified,” “non-naturally occurring,” “non-natural,” and“non-native” are interchangeable and indicate intentional humanmanipulation.

As used herein, “interrupted,” “broken,” and “discontinuous” are usedinterchangeably to mean a break in continuity, for example, in covalentbonds of a polynucleotide backbone. For example, a first polynucleotideand a second polynucleotide that are discontinuous each have a 5′terminus and a 3′ terminus (5′ terminus-first polynucleotide-3′ terminusand 5′ terminus-second polynucleotide-3′ terminus). For example, the 5′terminus of a DNA or RNA molecule is typically the fifth carbon in thesugar ring and the 3′ terminus is typically the hydroxyl group on thethird carbon in the sugar ring. Two polynucleotides each having a 5′terminus and a 3′ terminus are formed when the backbone of a singlepolynucleotide is broken at one site. A 5′ and/or 3′ terminus can becovalently modified, for example, by addition of a moiety (e.g., amoiety providing resistance to the degradative effects of exonucleases).

“Covalent bond,” “covalently attached,” “covalently bound,” “covalentlylinked,” “covalently connected,” and “molecular bond” are usedinterchangeably herein, and refer to a chemical bond that involves thesharing of electron pairs between atoms. Examples of covalent bondsinclude, but are not limited to, phosphodiester bonds andphosphorothioate bonds.

“Non-covalent bond,” “non-covalently attached,” “non-covalently bound,”“non-covalently linked,” “non-covalent interaction,” and “non-covalentlyconnected” are used interchangeably herein, and refer to any relativelyweak chemical bond that does not involve sharing of a pair of electrons.Multiple non-covalent bonds often stabilize the conformation ofmacromolecules and mediate specific interactions between molecules.Examples of non-covalent bonds include, but are not limited to hydrogenbonding, ionic interactions (e.g., Na⁺Cl⁻), van der Waals interactions,and hydrophobic bonds.

As used herein, “hydrogen bonding,” “hydrogen base pairing,” “hydrogenbond base pairing,” “hydrogen bonded,” and “hydrogen-bonded base pairs”are used interchangeably and refer to canonical hydrogen bonding andnon-canonical hydrogen bonding including, but not limited to,“Watson-Crick-hydrogen-bonded base pairs” (W—C-hydrogen-bonded basepairs or W—C hydrogen bonding); “Hoogsteen-hydrogen-bonded base pairs”(Hoogsteen hydrogen bonding); and “wobble-hydrogen-bonded base pairs”(wobble hydrogen bonding). W—C hydrogen bonding, including reverse W—Chydrogen bonding, refers to purine-pyrimidine base pairing, that isadenine:thymine, guanine:cytosine, and uracil: adenine. Hoogsteenhydrogen bonding, including reverse Hoogsteen hydrogen bonding, refersto a variation of base pairing in nucleic acids wherein two nucleobases,one on each strand, are held together by hydrogen bonds in the majorgroove. This non-W—C hydrogen bonding can allow a third strand to windaround a duplex and form triple-stranded helices. Wobble hydrogenbonding, including reverse wobble hydrogen bonding, refers to a pairingbetween two nucleotides in RNA molecules that does not followWatson-Crick base pair rules. There are four major wobble base pairs:guanine:uracil, inosine (hypoxanthine):uracil, inosine-adenine, andinosine-cytosine. Rules for canonical hydrogen bonding and non-canonicalhydrogen bonding are known to those of ordinary skill in the art (see,e.g., The RNA World, Third Edition (Cold Spring Harbor MonographSeries), R. F. Gesteland, Cold Spring Harbor Laboratory Press, ISBN978-0879697396 (2005); The RNA World, Second Edition (Cold Spring HarborMonograph Series), R. F. Gesteland, et al., Cold Spring HarborLaboratory Press, ISBN 978-0879695613 (1999); The RNA World (Cold SpringHarbor Monograph Series), R. F. Gesteland, et al., Cold Spring HarborLaboratory Press, ISBN 978-0879694562 (1993) (see, e.g., Appendix 1:Structures of Base Pairs Involving at Least Two Hydrogen Bonds, I.Tinoco); Principles of Nucleic Acid Structure, W. Saenger, SpringerInternational Publishing AG, ISBN 978-0-387-90761-1 (1988); Principlesof Nucleic Acid Structure, First Edition, S. Neidle, Academic Press,ISBN 978-01236950791 (2007)).

“Connect,” “connected,” and “connecting” are used interchangeablyherein, and refer to a covalent bond or a non-covalent bond between twomacromolecules (e.g., polynucleotides, proteins, and the like).

As used herein, “complementarity” refers to the ability of a nucleicacid sequence to form hydrogen bond(s) with another nucleic acidsequence (e.g., through canonical Watson-Crick base pairing). A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule that can form hydrogen bonds with a second nucleic acidsequence. If two polynucleotide sequences have 100% complementarity, thetwo sequences are perfectly complementary, i.e., all of the contiguousresidues of a first polynucleotide hydrogen bond with the same number ofcontiguous residues in a second polynucleotide.

As used herein, “binding” refers to a non-covalent interaction betweenmacromolecules (e.g., between a protein and a polynucleotide, between apolynucleotide and a polynucleotide, or between a protein and a protein,and the like). Such non-covalent interaction is also referred to as“associating” or “interacting” (e.g., if a first macromolecule interactswith a second macromolecule, the first macromolecule binds to secondmacromolecule in a non-covalent manner). Some portions of a bindinginteraction may be sequence-specific. “Sequence-specific binding,” asused herein, typically refers to one or more NASC polypeptidecompositions capable of forming a complex with one or more proteins(e.g., a Cas9 protein and/or a Cpf1 protein) to cause the proteins tobind a nucleic acid sequence (e.g., a DNA sequence) comprising a nucleicacid target sequence (e.g., a DNA target sequence) preferentiallyrelative to a second nucleic acid sequence (e.g., a second DNA sequence)without the nucleic acid target binding sequence (e.g., the DNA targetbinding sequence). All components of a binding interaction do not needto be sequence-specific, such as the protein binding with phosphateresidues in a DNA backbone. Binding interactions can be characterized bya dissociation constant (Kd). “Binding affinity” refers to the strengthof the binding interaction. An increased binding affinity is correlatedwith a lower Kd.

As used herein, a Cas protein (e.g., a Cas9 protein) is said to “target”a polynucleotide if a site-directed nucleoprotein complex comprising aCas protein binds or cleaves a polynucleotide at the nucleic acid targetsequence within the polynucleotide.

As used herein, “double-strand break” (DSB) refers to both strands of adouble-stranded segment of DNA being severed. In some instances, if sucha break occurs, one strand can be said to have a “sticky end” whereinnucleotides are exposed and not hydrogen bonded to nucleotides on theother strand. In other instances, a “blunt end” can occur wherein bothstrands remain fully base paired with each other.

“Donor polynucleotide,” “donor oligonucleotide,” and “donor template”are used interchangeably herein and can be a double-strandedpolynucleotide (e.g., a double-stranded DNA), a single-strandedpolynucleotide (e.g., single-stranded DNA), or a combination thereof.Donor polynucleotides comprise homology arms flanking the insertionsequence (e.g., DSBs in the DNA). The homology arms on each side canvary in length. Parameters for the design and construction of donorpolynucleotides are well-known in the art (see, e.g., Ran, F., et al.,Nature Protocols 8(11):2281-2308 (2013); Smithies, O., et al., Nature317:230-234 (1985); Thomas, K., et al., Cell 44:419-428 (1986); Wu, S.,et al., Nature Protocols 3:1056-1076 (2008); Singer, B., et al., Cell31:25-33 (1982); Shen, P., et al., Genetics 112:441-457 (1986); Watt,V., et al., Proceedings of the National Academy of Sciences of theUnited States of America 82:4768-4772 (1985); Sugawara, N., et al.,Journal of Molecular Cell Biology 12(2):563-575 (1992); Rubnitz, J., etal., Journal of Molecular Cell Biology 4(11):2253-2258 (1984); Ayares,D., et al., Proceedings of the National Academy of Sciences of theUnited States of America 83(14):5199-5203 (1986); Liskay, R, et al.,Genetics 115(1):161-167 (1987)).

As used herein, “homology-directed repair” (HDR) refers to DNA repairthat takes place in cells, for example, during repair of a DSB in DNA.HDR requires nucleotide sequence homology and uses a donorpolynucleotide to repair the sequence wherein the DSB (e.g., within aDNA target sequence) occurred. The donor polynucleotide generally hasthe requisite sequence homology with the sequence flanking the DSB sothat the donor polynucleotide can serve as a suitable template forrepair. HDR results in the transfer of genetic information from, forexample, the donor polynucleotide to the DNA target sequence. HDR mayresult in alteration of the DNA target sequence (e.g., insertion,deletion, or mutation) if the donor polynucleotide sequence differs fromthe DNA target sequence and part or all of the donor polynucleotide isincorporated into the DNA target sequence. In some embodiments, anentire donor polynucleotide, a portion of the donor polynucleotide, or acopy of the donor polynucleotide is integrated at the site of the DNAtarget sequence. For example, a donor polynucleotide can be used forrepair of the break in the DNA target sequence, wherein the repairresults in the transfer of genetic information (i.e., polynucleotidesequences) from the donor polynucleotide at the site or in closeproximity of the break in the DNA. Accordingly, new genetic information(i.e., polynucleotide sequences) may be inserted or copied at a DNAtarget sequence.

A “genomic region” is a segment of a chromosome in the genome of a hostcell that is present on either side of the nucleic acid target sequencesite or, alternatively, also includes a portion of the nucleic acidtarget sequence site. The homology arms of the donor polynucleotide havesufficient homology to undergo homologous recombination with thecorresponding genomic regions. In some embodiments, the homology arms ofthe donor polynucleotide share significant sequence homology to thegenomic region immediately flanking the nucleic acid target sequencesite; it is recognized that the homology arms can be designed to havesufficient homology to genomic regions farther from the nucleic acidtarget sequence site.

As used herein, “non-homologous end joining” (NHEJ) refers to the repairof a DSB in DNA by direct ligation of one end of the break to the otherend of the break without a requirement for a donor polynucleotide. NHEJis a DNA repair pathway available to cells to repair DNA without the useof a repair template. NHEJ in the absence of a donor polynucleotideoften results in nucleotides being randomly inserted or deleted at thesite of the DSB.

“Microhomology-mediated end joining” (MMEJ) is pathway for repairing aDSB in DNA. MMEJ involves deletions flanking a DSB and alignment ofmicrohomologous sequences internal to the broken ends before joining.MMEJ is genetically defined and requires the activity of, for example,CtIP, Poly(ADP-Ribose) Polymerase 1 (PARP1), DNA polymerase theta (Polθ), DNA Ligase 1 (Lig 1), or DNA Ligase 3 (Lig 3). Additional geneticcomponents are known in the art (see, e.g., Sfeir, A., et al., Trends inBiochemical Sciences 40:701-714 (2015)).

As used herein, “DNA repair” encompasses any process whereby cellularmachinery repairs damage to a DNA molecule contained in the cell. Thedamage repaired can include single-strand breaks or double-strandbreaks. At least three mechanisms exist to repair DSBs: HDR, NHEJ, andMMEJ. “DNA repair” is also used herein to refer to DNA repair resultingfrom human manipulation, wherein a target locus is modified, e.g., byinserting, deleting, or substituting nucleotides, all of which representforms of genome editing.

As used herein, “recombination” refers to a process of exchange ofgenetic information between two polynucleotides.

As used herein, the terms “regulatory sequences,” “regulatory elements,”and “control elements” are interchangeable and refer to polynucleotidesequences that are upstream (5′ non-coding sequences), within, ordownstream (3′ non-translated sequences) of a polynucleotide target tobe expressed. Regulatory sequences influence, for example, the timing oftranscription, amount or level of transcription, RNA processing orstability, and/or translation of the related structural nucleotidesequence. Regulatory sequences may include activator binding sequences,enhancers, introns, polyadenylation recognition sequences, promoters,transcription start sites, repressor binding sequences, stem-loopstructures, translational initiation sequences, internal ribosome entrysites (IRES), translation leader sequences, transcription terminationsequences (e.g., polyadenylation signals and poly-U sequences),translation termination sequences, primer binding sites, and the like.

Regulatory elements include those that direct constitutive, inducible,and repressible expression of a nucleotide sequence in many types ofhost cells and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).In some embodiments, a vector comprises one or more pol III promoters,one or more pol II promoters, one or more pol I promoters, orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer; see, e.g., Boshart, M., etal., Cell 41:521-530 (1985)), the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. It will be appreciated by thoseskilled in the art that the design of an expression vector may depend onsuch factors as the choice of the host cell to be transformed, the levelof expression desired, and the like. A vector can be introduced intohost cells to thereby produce transcripts, proteins, or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein.

“Gene,” as used herein, refers to a polynucleotide sequence comprisingexon(s) and related regulatory sequences. A gene may further compriseintron(s) and/or untranslated region(s) (UTR(s)).

As used herein, the term “operably linked” refers to polynucleotidesequences or amino acid sequences placed into a functional relationshipwith one another. For example, regulatory sequences (e.g., a promoter orenhancer) are “operably linked” to a polynucleotide encoding a geneproduct if the regulatory sequences regulate or contribute to themodulation of the transcription of the polynucleotide. Operably linkedregulatory elements are typically contiguous with the coding sequence.However, enhancers can function if separated from a promoter by up toseveral kilobases or more. Accordingly, some regulatory elements may beoperably linked to a polynucleotide sequence but not contiguous with thepolynucleotide sequence. Similarly, translational regulatory elementscontribute to the modulation of protein expression from apolynucleotide.

As used herein, “expression” refers to transcription of a polynucleotidefrom a DNA template, resulting in, for example, a messenger RNA (mRNA)or other RNA transcript (e.g., non-coding, such as structural orscaffolding RNAs). The term further refers to the process through whichtranscribed mRNA is translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be referred to collectively as“gene product(s).” Expression may include splicing the mRNA in aeukaryotic cell, if the polynucleotide is derived from genomic DNA(gDNA).

As used herein, the term “modulate” refers to a change in the quantity,degree or amount of a function. For example, a NASC polynucleotidecomposition/first nucleic acid binding protein/second nucleic acidbinding protein (e.g., Class 2 CRISPR-Cas proteins) complex, asdisclosed herein, may modulate the activity of a promoter sequence bybinding to two or more a nucleic acid target sequences at or near thepromoter. Depending on the action occurring after binding, the NASCpolynucleotide composition/first nucleic acid binding protein/secondnucleic acid binding protein complex can induce, enhance, suppress, orinhibit transcription of a gene operatively linked to the promotersequence. Thus, “modulation” of gene expression includes both geneactivation and gene repression.

Modulation can be assayed by determining any characteristic directly orindirectly affected by the expression of the target gene. Suchcharacteristics include, e.g., changes in RNA or protein levels, proteinactivity, product levels, expression of the gene, or activity level ofreporter genes. Accordingly, the terms “modulating expression,”“inhibiting expression,” and “activating expression” of a gene can referto the ability of a NASC polypeptide composition/nucleic acid bindingprotein(s) complex to change, activate, or inhibit transcription of agene.

“Vector” and “plasmid,” as used herein, refer to a polynucleotidevehicle to introduce genetic material into a cell. Vectors can be linearor circular. Vectors can contain a replication sequence capable ofeffecting replication of the vector in a suitable host cell (i.e., anorigin of replication). Upon transformation of a suitable host, thevector can replicate and function independently of the host genome orintegrate into the host genome. Vector design depends, among otherthings, on the intended use and host cell for the vector, and the designof a vector of the invention for a particular use and host cell iswithin the level of skill in the art. The four major types of vectorsare plasmids, viral vectors, cosmids, and artificial chromosomes.Typically, vectors comprise an origin of replication, a multicloningsite, and/or a selectable marker. An expression vector typicallycomprises an expression cassette.

As used herein, “expression cassette” refers to a polynucleotideconstruct generated using recombinant methods or by synthetic means andcomprising regulatory sequences operably linked to a selectedpolynucleotide to facilitate expression of the selected polynucleotidein a host cell. For example, the regulatory sequences can facilitatetranscription of the selected polynucleotide in a host cell, ortranscription and translation of the selected polynucleotide in a hostcell. An expression cassette can, for example, be integrated in thegenome of a host cell or be present in a vector to form an expressionvector.

As used herein, a “targeting vector” is a recombinant DNA constructtypically comprising tailored DNA arms, homologous to gDNA, that flankelements of a target gene or nucleic acid target sequence (e.g., a DSB).A targeting vector can comprise a donor polynucleotide. Elements of thetarget gene can be modified in a number of ways including deletionsand/or insertions. A defective target gene can be replaced by afunctional target gene, or in the alternative a functional gene can beknocked out. Optionally, the donor polynucleotide of a targeting vectorcomprises a selection cassette comprising a selectable marker that isintroduced into the target gene. Targeting regions (i.e., nucleic acidtarget sequences) adjacent or within a target gene can be used to affectregulation of gene expression.

As used herein, the terms “nucleic acid,” “nucleic acid sequence,”“nucleotide sequence,” “oligonucleotide,” and “polynucleotide” areinterchangeable and refer to a polymeric form of nucleotides. Thenucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA),analogs thereof, or combinations thereof, and may be of any length.Polynucleotides may perform any function and may have any secondary andtertiary structures. The terms encompass known analogs of naturalnucleotides and nucleotides that are modified in the base, sugar and/orphosphate moieties. Analogs of a particular nucleotide have the samebase-pairing specificity (e.g., an analog of A base pairs with T). Apolynucleotide may comprise one modified nucleotide or multiple modifiednucleotides. Examples of modified nucleotides include fluorinatednucleotides, methylated nucleotides, and nucleotide analogs. Nucleotidestructure may be modified before or after a polymer is assembled.Following polymerization, polynucleotides may be additionally modifiedvia, for example, conjugation with a labeling component or targetbinding component. A nucleotide sequence may incorporate non-nucleotidecomponents. The terms also encompass nucleic acids comprising modifiedbackbone residues or linkages, that are synthetic, naturally occurring,and non-naturally occurring, and have similar binding properties as areference polynucleotide (e.g., DNA or RNA). Examples of such analogsinclude, but are not limited to, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), Locked Nucleic Acid(LNA™) (Exiqon, Inc., Woburn, Mass.) nucleosides, glycol nucleic acid,bridged nucleic acids, and morpholino structures.

Peptide-nucleic acids (PNAs) are synthetic homologs of nucleic acidswherein the polynucleotide phosphate-sugar backbone is replaced by aflexible pseudo-peptide polymer. Nucleobases are linked to the polymer.PNAs have the capacity to hybridize with high affinity and specificityto complementary sequences of RNA and DNA.

In phosphorothioate nucleic acids, the phosphorothioate (PS) bondsubstitutes a sulfur atom for a non-bridging oxygen in thepolynucleotide phosphate backbone. This modification makes theinternucleotide linkage resistant to nuclease degradation. In someembodiments, phosphorothioate bonds are introduced between the last 3 to5 nucleotides at the 5′ or 3′ end of a polynucleotide sequence toinhibit exonuclease degradation. Placement of phosphorothioate bondsthroughout an entire oligonucleotide helps reduce degradation byendonucleases as well.

Threose nucleic acid (TNA) is an artificial genetic polymer. Thebackbone structure of TNA comprises repeating threose sugars linked byphosphodiester bonds. TNA polymers are resistant to nucleasedegradation. TNA can self-assemble by base-pair hydrogen bonding intoduplex structures.

Linkage inversions can be introduced into polynucleotides through use of“reversed phosphoramidites” (see, e.g.,www.ucalgary.ca/dnalab/synthesis/-modifications/linkages). A 3′-3′linkage at a terminus of a polynucleotide stabilizes the polynucleotideto exonuclease degradation by creating an oligonucleotide having two5′-OH termini and no 3′-OH terminus. Typically, such polynucleotideshave phosphoramidite groups on the 5′-OH position and a dimethoxytrityl(DMT) protecting group on the 3′-OH position. Normally, the DMTprotecting group is on the 5′-OH and the phosphoramidite is on the3′-OH.

Polynucleotide sequences are displayed herein in the conventional 5′ to3′ orientation unless otherwise indicated.

As used herein, “sequence identity” generally refers to the percentidentity of nucleotide bases or amino acids comparing a firstpolynucleotide or polypeptide to a second polynucleotide or polypeptideusing algorithms having various weighting parameters. Sequence identitybetween two polynucleotides or two polypeptides can be determined usingsequence alignment by various methods and computer programs (e.g.,BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, and the like) available throughthe worldwide web at sites including, but not limited to, GENBANK(www.ncbi.nlm.nih.gov/genbank/) and EMBL-EBI (www.ebi.ac.uk.). Sequenceidentity between two polynucleotides or two polypeptide sequences isgenerally calculated using the standard default parameters of thevarious methods or computer programs. A high degree of sequenceidentity, as used herein, between two polynucleotides or twopolypeptides is typically between about 90% identity and 100% identity,for example, about 90% identity or higher, preferably about 95% identityor higher, more preferably about 98% identity or higher. A moderatedegree of sequence identity, as used herein, between two polynucleotidesor two polypeptides is typically between about 80% identity to about 85%identity, for example, about 80% identity or higher, preferably about85% identity. A low degree of sequence identity, as used herein, betweentwo polynucleotides or two polypeptides is typically between about 50%identity and 75% identity, for example, about 50% identity, preferablyabout 60% identity, more preferably about 75% identity. For example, aCas protein (e.g., a Cas9 comprising amino acid substitutions) can havea low degree of sequence identity, a moderate degree of sequenceidentity, or a high degree of sequence identity, over its length to areference Cas protein (e.g., a wild-type Cas9). As another example, aNATNA can have a low degree of sequence identity, a moderate degree ofsequence identity, or a high degree of sequence identity, over itslength compared to a reference wild-type polynucleotide that complexeswith the reference Cas protein (e.g., a sgRNA that forms a complex withCas9).

As used herein, “hybridization” or “hybridize” or “hybridizing” is theprocess of combining two complementary single-stranded DNA or RNAmolecules so as to form a single double-stranded molecule (DNA/DNA,DNA/RNA, RNA/RNA) through hydrogen base pairing. Hybridizationstringency is typically determined by the hybridization temperature andthe salt concentration of the hybridization buffer; e.g., hightemperature and low salt provide high stringency hybridizationconditions. Examples of salt concentration ranges and temperature rangesfor different hybridization conditions are as follows: high stringency,approximately 0.01M to approximately 0.05M salt, hybridizationtemperature 5° C. to 10° C. below T_(m); moderate stringency,approximately 0.16M to approximately 0.33M salt, hybridizationtemperature 20° C. to 29° C. below T_(m); and low stringency,approximately 0.33M to approximately 0.82M salt, hybridizationtemperature 40° C. to 48° C. below T_(m). T_(m) of duplex nucleic acidsis calculated by standard methods well-known in the art (see, e.g.,Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press: New York (1982); Casey, J., et al.,Nucleic Acids Research 4:1539-1552 (1977); Bodkin, D. K., et al.,Journal of Virological Methods 10(1):45-52 (1985); Wallace, R. B., etal., Nucleic Acids Research 9(4):879-894 (1981)). Algorithm predictiontools to estimate T_(m) are also widely available. High stringencyconditions for hybridization typically refer to conditions under which apolynucleotide complementary to a target sequence predominantlyhybridizes with the target sequence, and substantially does nothybridize to non-target sequences. Typically, hybridization conditionsare of moderate stringency, preferably high stringency.

As used herein, a “stem element” or “stem structure” refers to apolynucleotide comprising two strands that are known or predicted toform a double-stranded region (the “stem element”). A “stem-loopelement” or “stem-loop structure” refers to a stem structure wherein the3′ end of one strand is covalently bonded to the 5′ end of the secondstrand by a nucleotide sequence of typically single-stranded nucleotides(“a stem-loop element nucleotide sequence”). In some embodiments, theloop element comprises a loop element nucleotide sequence of betweenabout 3 and about 20 nucleotides in length, preferably between about 4and about 10 nucleotides in length. In preferred embodiments, a loopelement nucleotide sequence is a single-stranded nucleotide sequence ofunpaired nucleic acid bases that do not interact through hydrogen bondformation to create a stem element within the loop element nucleotidesequence. The term “hairpin element” is also used herein to refer tostem-loop structures. Such structures are well known in the art. Thebase pairing may be exact; however, as is known in the art, a stemelement does not require exact base pairing. Thus, the stem element mayinclude one or more base mismatches or non-paired bases.

A “linker element nucleotide sequence” and “linker nucleotide sequence”are used interchangeably herein and refer to a single-stranded sequenceof one or more nucleotides covalently attached to a 5′ end, a 3′ end, orto both the 5′ and 3′ ends of a first polynucleotide sequence, andtypically refer to a single-stranded nucleic acid sequence connecting afirst polynucleotide sequence with a second polynucleotide sequence. Inpreferred embodiments, the linker element nucleotide sequence is asingle-stranded nucleotide sequence of unpaired nucleic acid bases thatdo not interact through hydrogen bond formation to create a stem elementwithin the linker element nucleotide sequence. In some embodiments, alinker element nucleotide sequence is between about 1 and about 20nucleotides in length, preferably between about 2 and about 10nucleotides in length.

As used herein, the term “amino acid” refers to natural and synthetic(unnatural) amino acids, including amino acid analogs, modified aminoacids, peptidomimetics, glycine, and D or L optical isomers.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areinterchangeable and refer to polymers of amino acids. A polypeptide maybe of any length. It may be branched or linear, it may be interrupted bynon-amino acids, and it may comprise modified amino acids. The termsalso refer to an amino acid polymer that has been modified through, forexample, acetylation, disulfide bond formation, glycosylation,lipidation, phosphorylation, pegylation, biotinylation, cross-linking,and/or conjugation (e.g., with a labeling component or ligand).Polypeptide sequences are displayed herein in the conventionalN-terminal to C-terminal orientation, unless otherwise indicated.

Polypeptides and polynucleotides can be made using routine techniques inthe field of molecular biology (see, e.g., standard texts discussedabove). Furthermore, essentially any polypeptide or polynucleotide isavailable from commercial sources.

The terms “fusion protein” and “chimeric protein,” as used herein, referto a single protein created by joining two or more proteins, proteindomains, or protein fragments that do not naturally occur together in asingle protein. For example, a fusion protein can contain a first domainfrom a Cas9 protein and a second domain a Csy4 protein. The modificationto include such domains in fusion protein may confer additional activityon the modified site-directed polypeptides. These activities can includenuclease activity, methyltransferase activity, demethylase activity, DNArepair activity, DNA damage activity, deamination activity, dismutaseactivity, alkylation activity, depurination activity, oxidationactivity, pyrimidine dimer forming activity, integrase activity,transposase activity, recombinase activity, polymerase activity, ligaseactivity, helicase activity, photolyase activity, glycosylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity or demyristoylation activity) thatmodifies a polypeptide associated with nucleic acid target sequence(e.g., a histone). A fusion protein can also comprise epitope tags(e.g., histidine tags, FLAG® (Sigma Aldrich, St. Louis, Mo.) tags, Myctags), reporter protein sequences (e.g., glutathione-S-transferase,beta-galactosidase, luciferase, green fluorescent protein, cyanfluorescent protein, yellow fluorescent protein), and/or nucleic acidbinding domains (e.g., a DNA binding domain, an RNA binding domain). Afusion protein can also comprise activator domains (e.g., heat shocktranscription factors, NFKB activators) or repressor domains (e.g., aKRAB domain). As described by Lupo, A., et al., Current Genomics 14(4):268-278 (2013), the KRAB domain is a potent transcriptional repressionmodule and is located in the amino-terminal sequence of most C2H2 zincfinger proteins (see, e.g., Margolin, J., et al., Proceedings of theNational Academy of Sciences of the United States of America91:4509-4513 (1994); Witzgall, R., et al., Proceedings of the NationalAcademy of Sciences of the United States of America 91:4514-4518(1994)). The KRAB domain typically binds to co-repressor proteins and/ortranscription factors via protein-protein interactions, causingtranscriptional repression of genes to which KRAB zinc finger proteins(KRAB-ZFPs) bind (see, e.g., Friedman J.R., et al., Genes & Development10:2067-2678 (1996)). In some embodiments, linker nucleic acid sequencesare used to join the two or more proteins, protein domains, or proteinfragments.

A “moiety,” as used herein, refers to a portion of a molecule. A moietycan be a functional group or describe a portion of a molecule withmultiple functional groups (e.g., that share common structural aspects).The terms “moiety” and “functional group” are typically usedinterchangeably; however, a “functional group” can more specificallyrefer to a portion of a molecule that comprises some common chemicalbehavior. “Moiety” is often used as a structural description. In someembodiments, a 5′ terminus, a 3′ terminus, or a 5′ terminus and a 3′terminus (e.g., a non-native 5′ terminus and/or a non-native 3′ terminusin a first stem element).

The term “affinity tag,” as used herein, typically refers to one or moremoieties that increases the binding affinity of a polynucleotidecomponent of a NASC polynucleotide composition, for example, tofacilitate formation of a NASC complex. Some embodiments of the presentinvention use an “affinity sequence,” which is a polynucleotide sequencecomprising one or more affinity tags. In some embodiments of the presentinvention, a polynucleotide component further comprises an affinitysequence located at the 5′ end, the 3′ end, or located between the 5′end and the 3′ end. Some embodiments of the present invention introduceone or more affinity tags to the N-terminal of a Cas protein sequence(e.g., a Cas9 protein sequence), to the C-terminal of a Cas proteinsequence, to a position located between the N-terminal and C-terminal ofa Cas protein sequence, or to combinations thereof. In some embodimentsof the invention, the Cas-polypeptide is modified with an affinity tagor an affinity sequence. A wide variety of affinity tags are disclosedin U.S. Published Patent Application No. 2014-0315985, published 23 Oct.2014.

As used herein, a “cross-link” is a bond that links one polymer chain(e.g., a polynucleotide or polypeptide) to another. Such bonds can becovalent bonds or ionic bonds. In some embodiments, one polynucleotidecan be bound to another polynucleotide by cross linking thepolynucleotides. In other embodiments, a polynucleotide can be crosslinked to a polypeptide. In additional embodiments, a polypeptide can becross linked to a polypeptide.

The term “cross-linking moiety,” as used herein, typically refers to amoiety suitable to provide cross linking between polynucleotidecomponents of a NASC polynucleotide composition. A cross-linking moietyis another example of an affinity tag.

The terms “ligand” and “ligand-binding moiety,” as used herein, refer tomoieties that facilitate the binding of polynucleotide components toform a NASC polynucleotide composition. Ligands and ligand-bindingmoieties are paired affinity tags.

As used herein, a “host cell” generally refers to a biological cell. Acell is the basic structural, functional and/or biological unit of anorganism. A cell can originate from any organism having one or morecells. Examples of host cells include, but are not limited to, aprokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, acell of a single-cell eukaryotic organism, a protozoal cell, a cell froma plant (e.g., cells from plant crops (such as soy, tomatoes, sugarbeets, pumpkin, hay, cannabis, tobacco, plantains, yams, sweet potatoes,cassava, potatoes, wheat, sorghum, soybean, rice, corn, maize,oil-producing Brassica (e.g., oil-producing rapeseed and canola),cotton, sugar cane, sunflower, millet, and alfalfa), fruits, vegetables,grains, seeds, flowering plants, conifers, gymnosperms, ferns,clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g.,Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsisgaditana, Chlorella pyrenoidosa, Sargassum patens C. agardh, and thelike), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell or acell from a mushroom), an animal cell, a cell from an invertebrateanimal (e.g., fruit fly, cnidarian, echinoderm, nematode, and the like),a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird,or mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, arodent, a rat, a mouse, a non-human primate, a human, and the like).Furthermore, a cell can be a stem cell or a progenitor cell.

As used herein, “stem cell” refers to a cell that has the capacity forself-renewal, i.e., the ability to go through numerous cycles of celldivision while maintaining the undifferentiated state. Stem cells can betotipotent, pluripotent, multipotent, oligopotent, or unipotent. Stemcells can be embryonic, fetal, amniotic, adult, or induced pluripotentstem cells.

As used herein, “induced pluripotent stem cells” refers to a type ofpluripotent stem cell that is artificially derived from anon-pluripotent cell, typically an adult somatic cell, by inducingexpression of specific genes.

“Plant,” as used herein, refers to whole plants, plant organs, planttissues, germplasm, seeds, plant cells, and progeny of the same. Plantcells include, without limitation, cells from seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen and microspores. Plant partsinclude differentiated and undifferentiated tissues including, but notlimited to, roots, stems, shoots, leaves, pollens, seeds, tumor tissueand various forms of cells and culture (e.g., single cells, protoplasts,embryos, and callus tissue). The plant tissue may be in plant or in aplant organ, tissue or cell culture. “Plant organ” refers to planttissue or a group of tissues that constitute a morphologically andfunctionally distinct part of a plant.

“Subject,” as used herein, refers to any member of the phylum Chordata,including, without limitation, humans and other primates, includingnon-human primates such as rhesus macaques, chimpanzees and other monkeyand ape species; farm animals, such as cattle, sheep, pigs, goats andhorses; domestic mammals, such as dogs and cats; laboratory animals,including rabbits, mice, rats and guinea pigs; birds, includingdomestic, wild, and game birds, such as chickens, turkeys and othergallinaceous birds, ducks, and geese; and the like. The term does notdenote a particular age or gender. Thus, the term includes adult, young,and newborn individuals as well as male and female. In some embodiments,a host cell is derived from a subject (e.g., stem cells, progenitorcells, or tissue-specific cells). In some embodiments, the subject is anon-human subject.

As used herein, “transgenic organism” refers to an organism whose genomeis genetically modified. The term includes the progeny (any generation)of a transgenic organism, provided that the progeny has the geneticmodification.

As used herein, “isolated” can refer to a nucleic acid or polypeptidethat, by human intervention, exists apart from its native environmentand is therefore not a product of nature. An isolated nucleic acid orpolypeptide can exist in a purified form and/or can exist in anon-native environment such as, for example, in a recombinant cell.

In a general aspect of the present invention, a NASC polynucleotidecomposition comprises a repeat element 1 connected with a repeat element2, a nucleic acid binding protein binding element 1 and a nucleic acidbinding protein binding element 2, and a spacer element 1 and a spacerelement 2.

Repeat element 1 and repeat element two are typically connected bycovalent bonds, non-covalent bonds, or a combination of covalent andnon-covalent bonds. In some embodiments, repeat element 1 and repeatelement 2 are connected by hydrogen-bonded base pairs.

The NASC polynucleotide composition is capable of associating withnucleic acid binding protein(s) to form a nucleoprotein complex. In someembodiments, two or more nucleic acid binding proteins having similarstructural motifs and functional motifs are used to form nucleoproteincomplexes with NASC polynucleotide compositions. In preferredembodiments, the nucleic acid binding proteins are Class 2 CRISPR-Casproteins. In some embodiments, the nucleic acid binding protein binds adouble-stranded nucleic acid binding protein binding sequence (“adouble-stranded nucleic acid binding protein”).

A NASC polynucleotide composition/nucleic acid binding protein 1/nucleicacid binding protein 2 complex is capable of preferentially binding anucleic acid target sequence in a polynucleotide (relative to apolynucleotide that does not comprise the nucleic acid target sequence).

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H,FIG. 4I, FIG. 4J, FIG. 4K, FIG. 4L, FIG. 4M, FIG. 4N, and FIG. 4Oillustrate generic examples of different types of nucleic acid scaffoldsof the present invention. These figures present relative locations ofdifferent elements in engineered nucleic acid sequences for formingscaffolds.

In some embodiments, a complex of two or more engineered nucleic acidsequences forming a scaffold comprises:

a first engineered nucleic acid comprising (i) a nucleic acid bindingprotein binding element 1 comprising a nucleic acid binding proteinbinding sequence 1 (e.g., a double-stranded nucleic acid binding proteinbinding sequence 1) having a first end and a second end, (ii) a repeatelement 1 comprising a repeat nucleic acid sequence 1 having a first endand a second end, and (iii) a spacer element 1 comprising a nucleic acidtarget binding sequence 1; and

a second engineered nucleic acid comprising (i) a nucleic acid bindingprotein binding element 1C comprising a nucleic acid binding proteinbinding sequence 2 (e.g., a double-stranded nucleic acid binding proteinbinding sequence 2) having a first end and a second end, (ii) a repeatelement 2 comprising a repeat nucleic acid sequence 2 having a first endand a second end, and (iii) a spacer element 2 comprising a nucleic acidtarget binding sequence 2.

Repeat nucleic acid sequence 1 and repeat nucleic acid sequence 1C arecomplementary. Repeat nucleic acid sequence 1C is also referred to as arepeat nucleic acid sequence 2. The repeat nucleic acid sequence 1 isconnected with the repeat nucleic acid sequence 1C throughhydrogen-bonded base pairs.

Table 2 presents a series of indicators used consistently in FIG. 4Athrough 4O.

TABLE 2 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region Nucleic Example of acid elementsequence General element Element comprises component a first engineerednucleic acid 1-1 a repeat a repeat nucleic a repeat nucleic element 1acid sequence 1 acid sequence 1-2 a nucleic acid a nucleic acid adouble-stranded binding protein binding protein nucleic acid bindingelement 1 binding sequence 1 binding protein binding sequence 1 1-3 aspacer a nucleic acid a nucleic acid element 1 sequence 1 target bindingsequence 1 a second engineered nucleic acid 2-1 a repeat a repeatnucleic a repeat nucleic element 2 acid sequence 2 acid sequence 2-2 anucleic acid a nucleic acid a double-stranded binding protein bindingprotein nucleic acid binding element 2 binding sequence 2 bindingprotein binding sequence 2 2-3 a spacer a nucleic acid a nucleic acidelement 2 sequence 2 target binding sequence 2 a third engineerednucleic acid 3-1 a repeat a repeat nucleic a repeat nucleic element 3acid sequence 3 acid sequence 3-2 a nucleic acid a nucleic acid adouble-stranded binding protein binding protein nucleic acid bindingelement 3 binding sequence 3 binding protein binding sequence 3 3-3 aspacer a nucleic acid a nucleic acid element 3 sequence 3 target bindingsequence 3 additional engineered nucleic acids # -1 a repeat a repeatnucleic a repeat nucleic element # acid sequence # acid sequence # -2 anucleic acid a nucleic acid a double-stranded binding protein bindingprotein nucleic acid binding element # binding sequence # bindingprotein binding sequence # # -3 a spacer a nucleic acid a nucleic acidelement # sequence # target binding sequence # An arrow in the figurecorresponds to a site that can comprise additional nucleic acidsequences, such as a linker element nucleic acid sequence, and a pair ofarrows illustrates boundaries of a particular element (e.g., the arrowsflanking region 1-1 in FIG. 4A). # = for additional engineered nucleicacids, sequential numbering following the number 3

FIG. 4A, FIG. 4C, FIG. 4E, FIG. 4G, FIG. 4I, and FIG. 4K, each presentsone example from a collection of six different arrangements of region1-1, region 1-2, and region 1-3 within a first engineered nucleic acid,and region 2-1, region 2-2, and region 2-3 within a second engineerednucleic acid, wherein the repeat nucleic acid sequence 1-1 is associatedwith the repeat nucleic acid sequence 2-1 through hydrogen bondingbetween the repeat nucleic acid sequence 1-1 and the repeat nucleic acidsequence 2-1. In these figures, the first engineered nucleic acid is asingle polynucleotide and the second engineered nucleic acid is a singlepolynucleotide. Each polynucleotide has a first end and a second end. Insome embodiments, the first end is a 5′ end and the second end is a 3′end. In other embodiments, the first end is a 3′ end and the second endis a 5′ end.

FIG. 4B, FIG. 4D, FIG. 4F, FIG. 4H, FIG. 4J, and FIG. 4L each presentsthe same arrangement as FIG. 4A, FIG. 4C, FIG. 4E, FIG. 4G, FIG. 4I, andFIG. 4K, respectively, wherein the first engineered nucleic acidcomprises multiple polynucleotides associated by hydrogen bonding(indicated in these figures as multiple straight lines between nucleicacid sequences) and the second engineered nucleic acid comprisesmultiple polynucleotides associated through hydrogen bonding. Eachpolynucleotide has a first end and a second end. In some embodiments,the first end is a 5′ end and the second end is a 3′ end, whereinstandard 5′ to 3′ orientation among the polynucleotides is maintained.In other embodiments, the first end is a 3′ end and the second end is a5′ end, wherein standard 5′ to 3′ orientation among the polynucleotidesis maintained.

FIG. 4M illustrates an example of a complex of three engineered nucleicacid sequences forming a scaffold. In this figure, the first, second,and third engineered nucleic acids are each a single polynucleotide. Thefirst and second engineered nucleic acids correspond to the first andsecond engineered nucleic acids presented in FIG. 4A, and the thirdengineered nucleic acid corresponds to the second engineered nucleicacid of FIG. 4G.

FIG. 4N illustrates an example of a complex of three engineered nucleicacid sequences forming a scaffold. In this figure, the first and thirdengineered nucleic acids are each a single polynucleotide. The secondengineered nucleic acid comprises multiple polynucleotides associatedthrough hydrogen bonding. The first engineered nucleic acid correspondsto the first engineered nucleic acid presented in FIG. 4C. The secondengineered nucleic acid corresponds to the second engineered nucleicacid presented in FIG. 4D. The third engineered nucleic acid correspondsto the second engineered nucleic acid of FIG. 4E.

FIG. 4O illustrates an example of a complex of three engineered nucleicacid sequences forming a scaffold. In this figure, the first and thirdengineered nucleic acids are each a single polynucleotide. The secondengineered nucleic acid comprises multiple polynucleotides associatedthrough hydrogen bonding. The first engineered nucleic acid correspondsto the first engineered nucleic acid presented in FIG. 4I. The secondengineered nucleic acid corresponds to the second engineered nucleicacid presented in FIG. 4F. The third engineered nucleic acid correspondsto the second engineered nucleic acid of FIG. 4K.

The present invention comprises a wide variety of nucleic acid-basedscaffolds that are composed of a complex of two or more engineerednucleic acid sequences. In preferred embodiments, the engineered nucleicacid sequences comprise elements of Class 2 CRISPR nucleic acidtargeting nucleic acids, for example, elements encoding nucleic acidsequences based on the sequences of Type 2-crRNAs, Type 2CRISPR-tracrRNAs, and Type 2 CRISPR single-guide RNAs. Examples of Class2 CRISPR-associated elements include, but are not limited to, theelements presented in FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 2A, FIG.2B, FIG. 3A, and FIG. 3B.

In some embodiments, the nucleic acid scaffolds comprise nucleic acidprotein binding sequences including, but not limited to, thoseassociated with genome editing systems (e.g., zinc finger nucleases(ZFNs), transcription activator-like effector-based nucleases (TALENs),meganucleases, and CRISPR-Cas). Examples of nucleic acid protein bindingsequences include, but are not limited to, those associated with thefollowing nucleic acid binding proteins: Type 2 CRISPR nucleic acidbinding proteins (e.g., Cpf1 protein, dCpf1 protein (catalyticallyinactive), Cas9 protein, and/or dCas9 protein (catalytically inactive));Argonaute proteins; double-stranded nucleic acid binding proteins (e.g.,Csy4 protein and/or Csy4* protein (catalytically inactive); see, e.g.,Haurwitz, R., et al., Science 329(5997):1355-1358 (2010); Sternberg, S.,et al., RNA 18(4):661-672 (2012); U.S. Pat. No. 9,115,348);single-stranded RNA binding proteins (e.g., p19 siRNA Binding Protein);single-stranded DNA binding proteins (e.g., adenovirus DBP, ExtremeThermostable SSB (single-stranded DNA binding protein); double-strandedRNA binding proteins (e.g., DICER); double-stranded DNA binding proteins(e.g., ZFNs); and double-stranded RNA/DNA hybrids (e.g., RibonucleaseH); as well as catalytically inactive versions thereof In additionalembodiments, the nucleic acid scaffolds and the associated nucleic acidbinding proteins are in nucleic acid scaffold/nucleic acid bindingprotein complexes, for example, nucleoprotein complexes andribonucleoprotein complexes.

In some embodiments, each of the nucleic acid binding protein bindingsequences of 1-2, 2-2, and/or 3-2, is, for example, a double-strandedDNA binding protein binding sequence, a single-stranded DNA bindingprotein binding sequence, a double-stranded RNA binding protein bindingsequence, a single-stranded RNA binding protein binding sequence, or adouble-stranded DNA/RNA hybrid binding protein binding sequence. Inpreferred embodiments, the nucleic acid binding protein that binds thenucleic acid binding protein binding sequence is a Cas9 protein or aCpf1 protein.

In particular embodiments, each of the nucleic acid sequence 1-1,nucleic acid sequence 1-2, and/or nucleic acid sequence 1-3 comprises anucleic acid sequence that binds to a target nucleic acid sequence(e.g., a spacer element).

In a first aspect of the present invention, a NASC polynucleotidecomposition comprises a NASC-PC1 and NASC-PC2. A NASC-PC1/NASC-PC2complex comprises a repeat element 1 connected with a repeat element 2,a double-stranded nucleic acid binding protein binding element 1 and adouble-stranded nucleic acid binding protein binding element 2, and aspacer element 1 and a spacer element 2. The double-stranded nucleicacid binding proteins that are capable of binding the NASC are one ormore Class 2 Type V CRISPR-Cpf1 proteins.

The NASC polynucleotide composition is capable of associating with twoClass 2 Type V CRISPR-Cpf1 proteins to form a nucleoprotein complex. Insome embodiments, each of NASC-PC1 and NASC-PC2 of the NASCpolynucleotide compositions is capable of associating with two Class 2Type V CRISPR-Cpf1 proteins to form a nucleoprotein complex (e.g., FIG.5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G).

In the first aspect of the present invention, the repeat element 1comprises a repeat nucleic acid sequence 1, the repeat element 2comprises a repeat nucleic acid sequence 1C, the nucleic acid bindingprotein binding element 1 comprises a double-stranded nucleic acidbinding protein binding sequence 1, the nucleic acid binding proteinbinding element 2 comprises a double-stranded nucleic acid bindingprotein binding sequence 2, the spacer element 1 comprises a nucleicacid target binding sequence 1, and the spacer element 2 comprises anucleic acid target binding sequence 2.

The arrangements of the elements are typically as follows: (i) therepeat element 1 is 5′ of the nucleic acid binding protein bindingelement 1, the nucleic acid binding protein binding element 1 is 5′ ofthe spacer element 1, and the repeat element 2 is 5′ of the nucleic acidbinding protein binding element 2, and the nucleic acid binding proteinbinding element 2 is 5′ of the spacer element 2; or (ii) the nucleicacid binding protein binding element 1 is 5′ of the repeat element 1,the repeat element 1 is 5′ of the spacer element 1, the nucleic acidbinding protein binding element 2 is 5′ of the repeat element 2, and therepeat element 2 is 5′ of the spacer element 2; or (iii) the nucleicacid binding protein binding element 1 is 5′ of the spacer element 1,the spacer element 1 is 5′ of the repeat element 1, the nucleic acidbinding protein binding element 2 is 5′ of the spacer element 2, and thespacer element 2 is 5′ of the repeat element 2.

In some embodiments of the first aspect, (i) the nucleic acid bindingprotein binding element 1 comprises a first stem element nucleic acidsequence 1-1 and a first stem element nucleic acid sequence 1-2, and thefirst stem element nucleic acid sequence 1-1 and the first stem elementnucleic acid sequence 1-2 form a first stem element 1 throughhydrogen-bonded base pairs, and/or (ii) the nucleic acid binding proteinbinding element 2 comprises a first stem element nucleic acid sequence2-1 and a first stem element nucleic acid sequence 2-2, and the firststem element nucleic acid sequence 2-1 and the first stem elementnucleic acid sequence 2-2 form a first stem element 1 throughhydrogen-bonded base pairs (e.g., FIG. 5B, FIG. 5F, FIG. 5G). In furtherembodiments, the first stem element nucleic acid sequence 1-1 and thefirst stem element nucleic acid sequence 1-2 are connected by a loopelement nucleic acid sequence 1 to form a first stem-loop element 1,and/or the first stem element nucleic acid sequence 2-1 and the firststem element nucleic acid sequence 2-2 are connected by a loop elementnucleic acid sequence 2 to form a first stem-loop element 2 (e.g., FIG.5A, FIG. 5C, FIG. 5D, FIG. 5E).

In additional embodiments, the repeat nucleic acid sequence 1 isconnected with the repeat nucleic acid sequence 1C throughhydrogen-bonded base pairs between the repeat nucleic acid sequence 1and the repeat nucleic acid sequence 1C.

In other embodiments, the repeat nucleic acid sequence 1 furthercomprises an affinity tag 1 and the repeat nucleic acid sequence 2further comprises an affinity tag 2, and the affinity tag 1 is connectedwith affinity tag 2. For example, the repeat nucleic acid sequence 1further comprises an effector protein binding site nucleic acid sequence1 and the repeat nucleic acid sequence 2 further comprises an effectorprotein binding site nucleic acid sequence 2, and an effector bindingsite 1 is formed by hydrogen base-pair bonding between the effectorprotein binding site nucleic acid sequence 1 and the effector proteinbinding site nucleic acid sequence 2. One example of an effector bindingsite is a Csy4 protein binding site.

Table 3 presents a series of indicators used consistently in FIG. 5A,FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, and FIG.5I.

TABLE 3 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region 500-507 corresponds to a firstengineered nucleic acid sequence first engineered nucleic acid componenta nucleic acid binding protein binding element 1 a double-strandednucleic acid binding protein binding element 1 501-502 corresponds to afirst stem element nucleic acid sequence 1-1 501-520 corresponds to aClass 2 Type V CRISPR protein binding site half stem sequence 1-1a520-502 corresponds to a Class 2 Type V CRISPR protein binding site halfstem sequence 1-1b 502-503 corresponds to a loop element nucleic acidsequence 1 503-504 corresponds to a first stem element nucleic acidsequence 1-2 a repeat element 1¹ 504-507 corresponds to a repeat nucleicacid sequence 1 504-505 corresponds to a linker element nucleic acidsequence 1-1 505-516 corresponds to a repeat nucleic acid sequence 1a516-517 corresponds to a linker element nucleic acid sequence 1-2517-506 corresponds to a repeat nucleic acid sequence 1b 506-507corresponds to a linker element nucleic acid sequence 1-3 a spacerelement 1 501-500 corresponds to a nucleic acid target binding sequence1 508-515 corresponds to a second engineered nucleic acid sequence asecond engineered nucleic acid component a nucleic acid binding proteinbinding element 2 a double-stranded nucleic acid binding protein bindingelement 2 514-513 corresponds to a first stem element nucleic acidsequence 2-1 521-513 corresponds to a Class 2 Type V CRISPR proteinbinding site half stem sequence 2-1c 514-521 corresponds to a Class 2Type V CRISPR protein binding site half stem sequence 2-1b 513-512corresponds to a loop element nucleic acid sequence 2 512-511corresponds to a first stem element nucleic acid sequence 2-2 a repeatelement 2 511-508 corresponds to a repeat nucleic acid sequence 1C²510-511 corresponds to a linker element nucleic acid sequence 1C- 1519-510 corresponds to a repeat nucleic acid sequence 1bC 518-519corresponds to a linker element nucleic acid sequence 1C- 2 509-518corresponds to a repeat nucleic acid sequence 2a 508-509 corresponds toa linker element nucleic acid sequence 1C- 3 a spacer element 2 514-515corresponds to a nucleic acid target binding sequence 2 ¹= repeatelement can include an effector protein binding site ²= “C” indicates acomplementary sequence

FIG. 5A presents an example of two engineered nucleic acids forming ascaffold of the present invention (NASC-PC1 and NASC-PC2). In someembodiments, the engineered nucleic acids are Class 2 Type V CRISPRnucleic acid targeting nucleic acids, for example, a Cpf1 nucleic acidtargeting nucleic acid comprising a repeat nucleic acid sequencecovalently attached to the 5′ end of the Cpf1 nucleic acid targetingnucleic acid. FIG. 5A, 500 to 507, illustrates a first engineerednucleic acid that comprises a first nucleic acid binding Class 2 Type VCRISPR protein binding sequence (FIG. 5A, 504 to 501), a nucleic acidtarget binding sequence 1 (FIG. 5A, 500 to 501) that is located 3′ ofthe first nucleic acid binding Class 2 Type V CRISPR protein bindingsequence, and a first repeat sequence 1 (FIG. 5A, 504-507) that islocated 5′ of the first nucleic acid binding Class 2 Type V CRISPRprotein binding sequence. FIG. 5A, 508 to 515, illustrates a secondengineered nucleic acid that comprises a second nucleic acid bindingClass 2 Type V CRISPR protein binding sequence (FIG. 5A, 514 to 511), anucleic acid target binding sequence 2 (FIG. 5A, 515 to 514) that islocated 3′ of the second nucleic acid binding Class 2 Type V CRISPRprotein binding sequence, and a first repeat sequence 2 (FIG. 5A,511-508) that is located 5′ of the second nucleic acid binding Class 2Type V CRISPR protein binding sequence.

The first engineered nucleic acid and the second engineered nucleic acidcan comprise additional elements such as effector protein bindingsequences, for example, a double-stranded nucleic acid binding proteinbinding site (e.g., a Csy4 protein binding site) created by theassociation of the repeat nucleic acid sequence 1 (FIG. 5A, 505-506) andthe repeat nucleic acid sequence 1C (FIG. 5A, 509-510) through hydrogenbond interactions.

FIG. 5B illustrates a modification of the example shown in FIG. 5A,wherein the loop element nucleic acid sequence 1 (FIG. 5A, 502 to 503)of the first engineered nucleic acid and the loop element nucleic acidsequence 2 (FIG. 5A, 513 to 512) of the second engineered nucleic acidare absent.

FIG. 5C presents an example of two engineered nucleic acids forming ascaffold of the present invention (NASC-PC1 and NASC-PC2). In someembodiments, the engineered nucleic acids are Class 2 Type V CRISPRnucleic acid targeting nucleic acids, for example, a Cpf1 nucleic acidtargeting nucleic acid comprising a repeat nucleic acid sequencecovalently attached to the 3′ end of the Cpf1 nucleic acid targetingnucleic acid. FIG. 5C illustrates a modification of the engineerednucleic acids, wherein a repeat sequence is added to the 3′ end (FIG.5C, 500) of a first engineered nucleic acid (FIG. 5C, 500 to 507) and acomplementary repeat sequence is added to the 3′ end (FIG. 5C, 515) ofthe second engineered nucleic acid (FIG. 5C, 508 to 515). The repeatsequence of the first engineered nucleic acid and the complementaryrepeat sequence of the second nucleic acid interact through hydrogenbase-pair bonding.

FIG. 5D presents a modification of NASC-PC1 and NASC-PC2 depicted inFIG. 5A. In FIG. 5D the repeat nucleic acid sequence covalently attachedto the 5′ end of each Cpf1 nucleic acid targeting nucleic acid comprisestwo repeat elements separated by a linker element nucleic acid sequence,wherein only one of the two repeat elements of FIG. 5D, I, iscomplementary to and capable of forming hydrogen bonds with one of therepeat elements of FIG. 5D, II. FIG. 5D illustrates a version of twoengineered nucleic acid forming a scaffold, wherein the repeat sequence1b (FIG. 5D, 506-517) of the first engineered nucleic acid (FIG. 5D, 500to 507) is capable of hydrogen base-pair bonding with the complementaryrepeat sequence 1bC (FIG. 5D, 519 to 510) of the second engineerednucleic acid (FIG. 5D, 515 to 508), wherein the repeat sequence 1b ofthe first engineered nucleic acid and the complementary repeat sequence1bC of the second engineered nucleic acid interact through hydrogenbase-pair bonding.

FIG. 5E presents an example of four engineered nucleic acids forming ascaffold based on two sets of the two engineered nucleic acids of FIG.5D. In this FIG. 5E, I, and FIG. 5E, II, provide points of reference tofacilitate comparison to the two engineered nucleic acids shown in FIG.5D (i.e., FIG. 5D, I, and II). FIG. 5E illustrates a modified version ofthe example shown in FIG. 5D wherein a repeat element of the firstengineered nucleic acid (FIG. 5E, I; NASC-PC-1) interacts with a repeatelement of the second engineered nucleic acid (FIG. 5E, II; NASC-PC-2)through hydrogen base-pair bonding, and a repeat element of the secondengineered nucleic acid (FIG. 5E, II) interacts with a repeat element ofthe third engineered nucleic acid (FIG. 5E, III; NASC-PC-3) throughhydrogen base-pair bonding, and a repeat element of the third engineerednucleic acid (FIG. 5E, III) interacts with a repeat element of a fourthengineered nucleic acid (FIG. 5E, IV; NASC-PC-4) through hydrogenbase-pair bonding, and a repeat element of the fourth engineered nucleicacid (FIG. 5E, IV) interacts with a repeat element of the firstengineered nucleic acid (FIG. 5E, I) through hydrogen base-pair bonding.

FIG. 5F illustrates a modified version of the example shown in FIG. 5D,wherein the loop element nucleic acid sequences (FIG. 5D, 502 to 503 andFIG. 5D, 513 to 512) are not present in the first engineered nucleicacid sequence (FIG. 5F, VIII and FIG. 5F, V), the second engineerednucleic acid sequence (FIG. 5G, VI), the third engineered nucleic acidsequence (FIG. 5G, VI), and the fourth engineered nucleic acid sequence(FIG. 5G, VII).

FIG. 5G presents an example of four engineered nucleic acids forming ascaffold based on two sets of the two engineered nucleic acids of FIG.5F. In this FIG. 5G, V, and FIG. 5G, VIII, provide points of referenceto facilitate comparison to the two engineered nucleic acids shown inFIG. 5F (i.e., FIG. 5F, V and VIII). FIG. 5G illustrates a modifiedversion of the example shown in FIG. 5F wherein a repeat element of thefirst engineered nucleic acid (FIG. 5G, V; NASC-PC-1) interacts with arepeat element of the second engineered nucleic acid (FIG. 5G, VI;NASC-PC-2) through hydrogen base-pair bonding, and a repeat element ofthe second engineered nucleic acid (FIG. 5G, VI; NASC-PC-2) interactswith a repeat element of the third engineered nucleic acid (FIG. 5G,VII; NASC-PC-3) through hydrogen base-pair bonding, and a repeat elementof the third engineered nucleic acid (FIG. 5G, VII) interacts with arepeat element of a fourth engineered nucleic acid (FIG. 5G, VIII;NASC-PC-4) through hydrogen base-pair bonding, and a repeat element ofthe fourth engineered nucleic acid (FIG. 5G, VIII) interacts with arepeat element of the first engineered nucleic acid (FIG. 5G, V) throughhydrogen base-pair bonding.

In other embodiments of the first aspect of the present invention, anucleoprotein complex can be formed by a nucleic acid binding proteinbinding to a macromolecule comprising a nucleic acid target bindingsequence 1, a repeat nucleic acid sequence 1, a repeat nucleic acidsequence 2, and a nucleic acid target binding sequence 1 (e.g., FIG. 5H,FIG. 5I).

FIG. 5H illustrates a version of two engineered nucleic acids forming ascaffold, wherein a Class 2 Type V CRISPR protein binding site half stemsequence 1-1b (FIG. 5H, 520-502) of a first engineered nucleic acid IX(FIG. 5H, 500 to 502; NASC-PC1) is capable of hydrogen base-pair bondingwith the complementary Class 2 Type V CRISPR protein binding site halfstem sequence 2-1b (FIG. 5H, 514 to 521) of a second engineered nucleicacid X (FIG. 5H, 515 to 513; NASC-PC2), and wherein the Class 2 Type VCRISPR protein binding site half stem sequence 1-1b of the firstengineered nucleic acid and the complementary Class 2 Type V CRISPRprotein binding site half stem sequence 2-1b of the second engineerednucleic acid interact through hydrogen base-pair bonding. Sequencevariation between the half stem sequences that is sufficient to providesequence specific hybridization between specific pairs of half stemsequences is possible because Class 2 Type V CRISPR protein binding siterecognition is tolerant of such sequence variation, provided thesecondary structure is maintained.

FIG. 5I illustrates a modified version of the example shown in FIG. 5H,wherein a Class 2 Type V CRISPR protein binding site half stem sequenceof the first engineered nucleic acid (FIG. 5I, IX) interacts with aClass 2 Type V CRISPR protein binding site half stem sequence of thesecond engineered nucleic acid (FIG. 5I, X); a Class 2 Type V CRISPRprotein binding site half stem sequence of the second engineered nucleicacid (FIG. 5I, X) interacts with a Class 2 Type V CRISPR protein bindingsite half stem sequence of the third engineered nucleic acid (FIG. 5I,XI); a Class 2 Type V CRISPR protein binding site half stem sequence ofthe third engineered nucleic acid (FIG. 5I, XI) interacts with a Class 2Type V CRISPR protein binding site half stem sequence of the fourthengineered nucleic acid (FIG. 5I, XII); and a Class 2 Type V CRISPRprotein binding site half stem sequence of the fourth engineered nucleicacid (FIG. 5I, XII) interacts with the Class 2 Type V CRISPR proteinbinding site half stem sequence of the first engineered nucleic acid(FIG. 5I, IX).

In a second aspect of the present invention, a NASC polynucleotidecomposition comprises at least NASC-PC1 and NASC-PC2. ANASC-PC1/NASC-PC2 complex comprises a repeat element 1 connected to arepeat element 2, a double-stranded nucleic acid binding protein bindingelement 1 and a double-stranded nucleic acid binding protein bindingelement 2, and a spacer element 1 and a spacer element 2. Embodiments ofthe present invention include NASC polynucleotide composition comprisingdouble-stranded nucleic acid binding protein binding elementscorresponding to one or more Class 2 CRISPR-Cas proteins.

In some embodiments, the NASC polynucleotide composition is capable ofassociating with two Class 2 Type II CRISPR-Cas9 proteins to form anucleoprotein complex. FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG.6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6K, FIG. 6L, and FIG. 6M illustrateelements and examples of engineered nucleic acid scaffolds of thepresent invention typically comprising a nucleic acid binding Class 2CRISPR protein binding sequence.

Table 4 presents a series of indicators used consistently in FIG. 6A,FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G.

TABLE 4 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region a first engineered nucleic acidcomponent a first engineered nucleic acid sequence (corresponds to601-611) a nucleic acid binding protein binding element 1 adouble-stranded nucleic acid binding protein binding element 1 601-602corresponds a linker element nucleic acid sequence 602-603 correspondsto a hairpin nucleic acid sequence 1-2 603-604 corresponds to a linkerelement nucleic acid sequence 604-605 corresponds to a hairpin nucleicacid sequence 1-1 605-606 corresponds to a linker element nucleic acidsequence 606-607 corresponds to a nexus element nucleic acid sequence1-1 607-608 corresponds to a linker element nucleic acid sequence arepeat element 1¹ 608-609 corresponds to a repeat nucleic acid sequence1 608-623 corresponds to a linker element nucleic acid sequence 1-1623-624 corresponds to a repeat nucleic acid sequence 1a 623-631corresponds to a repeat nucleic acid sequence 1a1 631-632 corresponds toa bulge nucleic acid sequence 1a1 632-624 corresponds to a repeatnucleic acid sequence 1a2 624-625 corresponds to a linker elementnucleic acid sequence 1-2 624-647 corresponds to a linker elementnucleic acid sequence 1-2-1 647-648 corresponds to a repeat nucleic acidsequence 1-2a 648-625 corresponds to a linker element nucleic acidsequence 1-2-2 625-626 corresponds to a repeat nucleic acid sequence 1b625-633 corresponds to a repeat nucleic acid sequence 1b1 633-634corresponds to a bulge nucleic acid sequence 1b1 634-626 corresponds toa repeat nucleic acid sequence 1b2 626-609 corresponds to a linkerelement nucleic acid sequence 1-3 609-610 corresponds to a linkerelement nucleic acid sequence a spacer element 1 610-611 corresponds toa nucleic acid target binding sequence 1 a second engineered nucleicacid component a second engineered nucleic acid sequence (corresponds to612-622) a nucleic acid binding protein binding element 2 adouble-stranded nucleic acid binding protein binding element 2 612-613corresponds a linker element nucleic acid sequence 613-614 correspondsto a hairpin nucleic acid sequence 2-2 614-615 corresponds to a linkerelement nucleic acid sequence 615-616 corresponds to a hairpin nucleicacid sequence 2-1 616-617 corresponds to a linker element nucleic acidsequence 617-618 corresponds to a nexus element nucleic acid sequence2-1 618-619 corresponds to a linker element nucleic acid sequence arepeat element 2 619-620 corresponds to a repeat nucleic acid sequence1C² 619-627 corresponds to a linker element nucleic acid sequence 2-3627-628 corresponds to a repeat nucleic acid sequence 1bC 627-635corresponds to a repeat nucleic acid sequence 1b2C 635-636 correspondsto a bulge nucleic acid sequence 2b2 636-628 corresponds to a repeatnucleic acid sequence 1b1C 628-629 corresponds to a linker elementnucleic acid sequence 2-2 628-649 corresponds to a linker elementnucleic acid sequence 2-2-2 649-650 corresponds to a repeat nucleic acidsequence 1-2aC 650-629 corresponds to a linker element nucleic acidsequence 2-2-1 629-630 corresponds to a repeat nucleic acid sequence 1aC629-637 corresponds to a repeat nucleic acid sequence 1a2C 637-638corresponds to a bulge nucleic acid sequence 2a2 638-630 corresponds toa repeat nucleic acid sequence 1a1C 630-620 corresponds to a linkerelement nucleic acid sequence 2-1 620-621 corresponds to a linkerelement nucleic acid sequence a spacer element 2 621-622 corresponds toa nucleic acid target binding sequence 2 ¹= repeat element can includean effector protein binding site ²= “C” indicates a complementarysequence

Each of a first, a second and a third element can comprise additionalnucleic acid sequences, for example, 5′ of the element, 3′ of theelement, or both 5′ of the element and 3′ of the element.

Each of a first, a second and a third element can comprise additionalnucleic acid sequences, for example, 5′ of the element, 3′ of theelement, or both 5′ of the element and 3′ of the element.

FIG. 6A, 601-611, illustrates an example of first engineered nucleicacid that comprises a first element comprising a Class 2 Type II CRISPRbinding protein sequence (FIG. 6A, 601-607), a second element comprisinga repeat nucleic acid sequence 1 (FIG. 6A, 608-609), and a third elementcomprising a nucleic acid sequence 1 (FIG. 6A, 610-611). No nucleic acidsequence within the repeat nucleic acid sequence 1 associates with anynucleic acid sequence within the repeat nucleic acid sequence 1 to forma stem element through hydrogen bonding capable of binding to a Class 2Type II CRISPR-Cas protein.

FIG. 6B illustrates a modification to FIG. 6A, wherein the firstengineered nucleic acid (FIG. 6B, 601-611) is associated with the secondengineered nucleic acid (FIG. 6B, 612-622) through hydrogen base-pairbonding between the repeat nucleic acid sequence 1 (FIG. 6A, 608-609)and the repeat nucleic acid sequence 1C (FIG. 6A, 619-620).

A NASC polynucleotide composition similar to the composition illustratedin FIG. 6B can be constructed for use with Class 2 Type V CRISPR-Casproteins to form a nucleoprotein complex. An example of this type ofNASC polynucleotide composition is illustrated in FIG. 10, V.

FIG. 6C illustrates an example of a first engineered nucleic acid,wherein the second element further comprises, in a 3′ to 5′ orientation,a linker element nucleic acid sequence 1-1 (FIG. 6C, 608-623), a repeatnucleic acid sequence 1a (FIG. 6C, 623-624), a linker element nucleicacid sequence 1-2 (FIG. 6C, 624-625), a repeat nucleic acid sequence 1b(FIG. 6D, 625-626), and a linker element nucleic acid sequence 1-3 (FIG.6C, 626-609). No nucleic acid sequence within the repeat nucleic acidsequence 1 associates with any nucleic acid sequence within the repeatnucleic acid sequence 1 to form a stem element through hydrogen bondingcapable of binding to a Class 2 Type II CRISPR-Cas protein.

FIG. 6D illustrates a modification to FIG. 6C in which two engineerednucleic acids form a scaffold, wherein the first engineered nucleic acidis associated with the second engineered nucleic acid through hydrogenbase-pair bonding between the repeat nucleic acid sequence 1a (FIG. 6D,623-624) and the repeat nucleic acid sequence 1aC (FIG. 6D, 629-630) andthrough hydrogen base-pair bonding between the repeat nucleic acidsequence 1b (FIG. 6D, 625-626) and the repeat nucleic acid sequence 1bC(FIG. 6D, 627-628).

FIG. 6E, illustrates an example of a first engineered nucleic acid,wherein the second element further comprises, in a 3′ to 5′ orientation,a linker element nucleic acid sequence 1-1 (FIG. 6C, 608-623), a repeatnucleic acid sequence 1a1 (FIG. 6C, 623-631), a bulge nucleic acidsequence (FIG. 6E, 631-632), a repeat nucleic acid sequence 1a2 (FIG.6E, 632-624), a linker element nucleic acid sequence 1-2 (FIG. 6C,624-625), a repeat nucleic acid sequence 1b1 (FIG. 6D, 625-633), a bulgenucleic acid sequence 1b1 (FIG. 6E, 633-634), and a repeat nucleic acidssequence 1b2 (FIG. 6E, 634-626). No nucleic acid sequence within therepeat nucleic acid sequence 1 associates with any nucleic acid sequencewithin the repeat nucleic acid sequence 1 to form a stem element throughhydrogen bonding capable of binding to a Class 2 Type II CRISPR-Casprotein.

FIG. 6F illustrates a modification to FIG. 6E in which two engineerednucleic acids form a scaffold, wherein the first engineered nucleic acidis associated with the second engineered nucleic acid through hydrogenbase-pair bonding between the repeat nucleic acid sequence 1a1 (FIG. 6F,623-631) and the repeat nucleic acid sequence 1a1C (FIG. 6F, 638-630),through hydrogen base-pair bonding between the repeat nucleic acidsequence 1a2 (FIG. 6F, 632-624) and the repeat nucleic acid sequence1a2C (FIG. 6F, 629-637), through hydrogen base-pair bonding between therepeat nucleic acid sequence 1b1 (FIG. 6F, 625-633) and the repeatnucleic acid sequence 1b1C (FIG. 6F, 636-628), and through hydrogenbase-pair bonding between the repeat nucleic acid sequence 1b2 (FIG. 6E,634-626) and repeat nucleic acid sequence 1b2C (FIG. 6E, 627-635).

FIG. 6G is a variation of the NASC polynucleotide compositionillustrated in FIG. 6F. The linker element nucleic acid sequence 1-2 ismodified by insertion of an effector protein binding site nucleic acidsequence 1 (FIG. 6G, 647-648) and the linker element nucleic acidsequence 2-2 is modified by insertion of an effector protein bindingsite nucleic acid sequence 2 (FIG. 6G, 649-650). The effector proteinbinding site nucleic acid sequence 1 and the effector protein bindingsite nucleic acid sequence 2 connect to form an effector protein bindingsite through hydrogen-bonded base pairs. In one embodiment, the effectorprotein binding site is a Csy4 binding site. An enzymatically inactiveform of the Csy4 protein can bind the site to further stabilize the NASCpolynucleotide composition structure. An enzymatically active form ofthe Csy4 protein can bind the site to destabilize (e.g., throughendoribonuclease activity) the NASC polynucleotide composition structure(e.g., to induce disruption of NASC polynucleotide composition/nucleicacid binding proteins-based closed cage structures). FIG. 6K illustratesa NASC polynucleotide composition capable of associating with a firstClass 2 Type II CRISPR-Cas9 ortholog protein and a second Class 2 TypeII CRISPR-Cas9 ortholog protein to form a nucleoprotein complex. FIG. 6Killustrates a NASC polynucleotide composition comprising a NASC-PC1 anda NASC-PC2. The NASC-PC1 (FIG. 6K, IX) comprises, in a 5′ to 3′direction: a nucleic acid target binding sequence 1; a linker nucleicacid sequence 1 comprising, a repeat nucleic acid sequence 1a, a repeatnucleic acid sequence 1b, a repeat nucleic acid 1c, and a repeat nucleicacid 1d; and S. thermophilus Class 2 Type II CRISPR-Cas9 nucleic acidbinding protein binding sequence 1. The NASC-PC2 (FIG. 6K, X) comprises,in a 5′ to 3′ direction: a nucleic acid target binding sequence 2; alinker nucleic acid sequence 1 comprising, a repeat nucleic acidsequence 1dC, a repeat nucleic acid sequence 1cC, a repeat nucleic acid1bC, and a repeat nucleic acid sequence 1aC; and a S. pyogenes Class 2Type II CRISPR-Cas9 nucleic acid binding protein binding sequence 1. TheNASC-PC1 and the NASC-PC2 connected through hydrogen-bonded base pairsbetween the repeat nucleic acid sequence 1a/the repeat nucleic acidsequence 1aC, the repeat nucleic acid sequence 1b/the repeat nucleicacid sequence 1bC, the repeat nucleic acid sequence 1c/the repeatnucleic acid 1cC, and the repeat nucleic acid sequence 1d/the repeatnucleic acid 1dC to form a macromolecule. The macromolecule is capableof binding a S. thermophilus Class 2 Type II CRISPR-Cas9 protein (aroundthe repeat nucleic acid sequence 1d/the repeat nucleic acid sequence 1dCregion and the repeat nucleic acid sequence 1c/the repeat nucleic acidsequence 1cC region) and a S. pyogenes Class 2 Type II CRISPR-Cas9protein (around the repeat nucleic acid sequence 1b/the repeat nucleicacid sequence 1bC region, the repeat nucleic acid sequence 1a/the repeatnucleic acid sequence 1aC region). Use of such NASC polynucleotidecomposition/Cas9 ortholog protein complexes provides, for example, anincreased number of available target sequences in view of PAMvariability between the Cas9 ortholog proteins (versus use of one guidenucleic acid/Cas9 protein complex comprising either Cas9 orthologalone). Further, NASC polynucleotide composition/Cas9 ortholog proteincomplexes may improve specificity of targeting of a polynucleotideregion by providing greater flexibility in choosing nearby targetsequences in view of PAM variability between the Cas9 ortholog proteins(versus use of one guide nucleic acid/Cas9 protein complex comprisingeither Cas9 ortholog alone). In view of the teachings of the presentspecification, one of ordinary skill in the art can apply this use oftwo or more different Cas9 ortholog proteins by combining differentcomponents of the NASC polynucleotide compositions described herein.

In a further embodiment, the NASC-PC1 (FIG. 6K, IX) and NASC-PC2 (FIG.6K, X) are capable of associating with the same Class 2 Type IICRISPR-Cas9 ortholog protein (see, e.g., Fonfara, I., et al., NucleicAcids Research 42(4):2577-2590 (2014)). In this embodiment, the repeatnucleic acid sequence 1d/the repeat nucleic acid sequence 1dC region,and the repeat nucleic acid sequence 1c/the repeat nucleic acid sequence1cC region are capable of associating with a S. mutans Class 2 Type IICRISPR-Cas9 protein and are also capable of associating with a S.pyogenes Class 2 Type II CRISPR-Cas9 protein. The repeat nucleic acidsequence 1b/the repeat nucleic acid sequence 1bC region, and the repeatnucleic acid sequence 1a/the repeat nucleic acid sequence 1aC region arecapable of associating with a S. pyogenes Class 2 Type II CRISPR-Cas9protein and are also capable of associating with a S. mutans Class 2Type II CRISPR-Cas9 protein. For example, although the repeat regions ofNASC-PC1 (FIG. 6K, IX) and the NASC-PC2 (FIG. 6K, X) are derived fromdifferent species containing Class 2 Type II CRISPR loci (e.g., S.pyogenes or S. mutans), only one Class 2 Type II CRISPR-Cas9 protein(e.g., a S. pyogenes Class 2 Type II CRISPR-Cas9 protein or a S. mutansClass 2 Type II CRISPR-Cas9 protein) is used to form a NASCpolynucleotide composition/Cas9 complex. One advantage of this type ofNASC polynucleotide composition is the flexibility to use either of twoCas9 proteins with the same NASC polynucleotide composition, and each ofthe Cas9 proteins recognize different PAM sequences. Thus, the number ofpossible binding sites that can be targeted by the NASC polynucleotidecomposition is increased.

In some embodiments of the second aspect of the present invention, aNASC polynucleotide composition comprises at least threepolynucleotides, wherein the complex comprises a repeat element 1connected to a repeat element 1C, the repeat element 2 connected to arepeat element 2C, the repeat element 3 connected to a repeat element3C, a double-stranded nucleic acid binding protein binding element 1 aspacer element 1, a spacer element 2, and a spacer element 3, whereinthe NASC polynucleotide composition is capable of binding three nucleicacid binding proteins. In some embodiments, the nucleic acid bindingproteins are double-stranded nucleic acid binding proteins. In preferredembodiments, the nucleic acid binding proteins Class 2 CRISPR-Casproteins.

Table 5 presents a series of additional indicators used in FIG. 6H andFIG. 6I.

TABLE 5 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region a first engineered nucleic acidcomponent (I) a first engineered nucleic acid sequence a nucleic acidbinding protein binding element 1 a repeat element 1¹ 608-609corresponds to a repeat nucleic acid sequence 1 608-623 corresponds to alinker element nucleic acid sequence 1-1 623-624 corresponds to a repeatnucleic acid sequence 1a 624-625 corresponds to a linker element nucleicacid sequence 1-2 625-626 corresponds to a repeat nucleic acid sequence1b 626-609 corresponds to a linker element nucleic acid sequence 1-3 aspacer element 1 a second engineered nucleic acid component (II) asecond engineered nucleic acid sequence a nucleic acid binding proteinbinding element 2 a repeat element 2 619-653 corresponds to a repeatnucleic acid sequence 2 619-627 corresponds to a linker element nucleicacid sequence 2-3 627-628 corresponds to a repeat nucleic acid sequence1bC² 628-651 corresponds to a linker element nucleic acid sequence 2-2651-652 corresponds to a repeat nucleic acid sequence 2a 652-653corresponds to a linker element nucleic acid sequence 2-4 a spacerelement 2 a third engineered nucleic acid component (III) a thirdengineered nucleic acid sequence a nucleic acid binding protein bindingelement 3 a repeat element 3 654-659 corresponds to a repeat nucleicacid sequence 1 654-655 corresponds to a linker element nucleic acidsequence 3-3 655-656 corresponds to a repeat nucleic acid sequence 2aC656-657 corresponds to a linker element nucleic acid sequence 3-2657-658 corresponds to a repeat nucleic acid sequence 1aC 658-659corresponds to a linker element nucleic acid sequence 3-1 a spacerelement 3 ¹= repeat element can include an effector protein binding site²= “C” indicates a complementary sequence

FIG. 6H illustrates a modification to FIG. 6C where three engineerednucleic acids form a scaffold, wherein a first engineered nucleic acid(FIG. 6H, I) is associated with a second engineered nucleic acid (FIG.6H, II) through hydrogen base-pair bonding between repeat nucleic acidsequence 1b (FIG. 6H, 625-626) and repeat nucleic acid sequence 1bC(FIG. 6H, 627-628), and the second engineered nucleic acid (FIG. 6H, II)is associated with the third engineered nucleic acid (FIG. 6H, III)through hydrogen base-pair bonding between repeat nucleic acid sequence2a (FIG. 6H, 638-639) and repeat nucleic acid sequence 2aC (FIG. 6H,642-643), and the third engineered nucleic acid (FIG. 6H, III) isassociated with the first engineered nucleic acid (FIG. 6H, I) throughhydrogen base-pair bonding between repeat nucleic acid sequence 1aC(FIG. 6H, 644-645) and repeat nucleic acid sequence 1a (FIG. 6H,623-624).

FIG. 6I illustrates a modification to FIG. 6E where three engineerednucleic acids form a scaffold using the engineered nucleic aciddescribed in FIG. 6E, wherein the first engineered nucleic acid (FIG.6I, IV) is associated with the second engineered nucleic acid (FIG. 6I,V) through hydrogen base-pair bonding between repeat sequences, and thesecond engineered nucleic acid (FIG. 6I, V) is associated with the thirdengineered nucleic acid (FIG. 6I, VI) through hydrogen base-pair bondingbetween repeat sequences, and the third engineered nucleic acid (FIG.6I, VI) is associated with the first engineered nucleic acid (FIG. 6I,IV) through hydrogen base-pair bonding between repeat sequences.

FIG. 6J illustrates a NASC polynucleotide composition capable ofassociating with a Class 2 Type II CRISPR-Cas protein and a Class 2 TypeV CRISPR-Cpf1 protein to form a nucleoprotein complex.

FIG. 6J illustrates a NASC polynucleotide composition capable ofassociating with a Class 2 Type II CRISPR-Cas9 protein and a Class 2Type V CRISPR-Cpf1 protein to form a nucleoprotein complex. FIG. 6Jillustrates a NASC polynucleotide composition comprising a NASC-PC1comprising a spacer element 1 and a spacer element 2 (NASC-PC-2TS; FIG.6J, VII). The NASC-PC-2TS comprising, in a 5′ to 3′ direction: a Class 2Type II CRISPR-Cas9 nucleic acid target binding sequence 1; a linkernucleic acid sequence 1 comprising a repeat nucleic acid sequence 1a, arepeat nucleic acid sequence 1b, and a repeat nucleic acid sequence 1c;and a Class 2 Type V CRISPR-Cpf1 nucleic acid target binding sequence 1.The NASC polynucleotide composition further comprises a NASC-PC2comprising a concatenate comprising a Class 2 Type II CRISPR-Cas9nucleic acid binding protein binding sequence 1 and a Class 2 Type VCRISPR-Cpf1 nucleic acid binding protein binding sequence 2 (NASC-PC-CE;FIG. 6J, VIII). The NASC-PC-CE further comprises a repeat nucleic acidsequence 1aC, a repeat nucleic acid sequence 1bC, and a repeat nucleicacid sequence 1cC through which the NASC-PC-CE is connected to theNASC-PC-2TS through hydrogen-bonded base pairs to form a macromoleculethat is capable of binding a Class 2 Type II CRISPR-Cas9 protein and aClass 2 Type V CRISPR-Cpf1 protein. Use of such NASC polynucleotidecomposition/Cas9 protein/Cpf1 protein complexes provides, for example,an increased number of available target sequences in view of PAMvariability and target sequence length differences between the Cas9protein and the Cpf1 protein (versus use of one guide nucleic acid/Casprotein complex comprising either Cas9 protein or Cpf1 protein alone).Furthermore, NASC polynucleotide composition/Cas9 protein/Cpf1 proteincomplexes may improve specificity of targeting of a polynucleotideregion by providing greater flexibility in choosing nearby targetsequences in view of PAM variability and target sequence lengths betweenthe Cas9 protein and the Cpf1 protein (versus use of one guide nucleicacid/Cas protein complex comprising either Cas9 protein or Cpf1 proteinalone). In view of the teachings of the present specification, one ofordinary skill in the art can apply this use of two or more differentCas proteins by combining different components of the NASCpolynucleotide compositions described herein.

In other embodiments, a first repeat nucleic acid sequence of a pairfurther comprises a first affinity tag and a second repeat nucleic acidsequence of the pair further comprises a second affinity tag, and thefirst affinity tag is connected with the second affinity tag. Forexample, the repeat nucleic acid sequence 1 further comprises aneffector protein binding site nucleic acid sequence 1 and the repeatnucleic acid sequence 2 further comprises an effector protein bindingsite nucleic acid sequence 2, and an effector binding site 1 is formedby hydrogen base-pair bonding between the effector protein binding sitenucleic acid sequence 1 and the effector protein binding site nucleicacid sequence 2. One example of an effector binding site is a Csy4protein binding site.

In a third aspect of the present invention, NASC polynucleotidecomposition comprises an engineered concatenated nucleic acid component(“NASC-PC-CT”) and at least a NASC-PC1 and a NASC-PC2.

In one embodiment of the third aspect of the present invention, anengineered NASC polynucleotide concatenated element (NASC-PC-CE)comprises, in a 3′ to 5′ direction: a first concatenate element 1comprising a nucleic acid binding protein binding element 1, and asecond concatenate element 1 comprising a repeat element A1, wherein therepeat element A1 comprises a repeat nucleic acid sequence A1; a firstconcatenate element 2 comprising a nucleic acid binding protein bindingelement 2; and a second concatenate element 2 comprising a repeatelement 2, wherein the repeat element 2 comprises a repeat nucleic acidsequence A2. The first concatenate element 1 is connected to the secondconcatenate element 1, the second concatenate element 1 is connected tothe first concatenate element 2, and the first concatenate element 2 isconnected to the second concatenate element 2 to form the NASC-PC-CE.

A third concatenate element 1 (NASC-PC-CE3-1) comprises, in a 3′ to 5′direction a repeat element A1C comprising a repeat nucleic acid sequenceA1C, and a spacer element 1 comprising a nucleic acid target bindingsequence 1. A third concatenate element 2 (NASC-PC-CE3-2) comprises, ina 3′ to 5′ direction a repeat element A2C comprising a repeat nucleicacid sequence A2C, and a spacer element 2 comprising a nucleic acidtarget binding sequence 2. The repeat nucleic acid sequence A1 isconnected with the repeat nucleic acid sequence A1-C, the repeat nucleicacid sequence A2 is connected with the repeat nucleic acid sequence A2-Cto form the NASC-PC-CE.

A first nucleic acid binding protein is capable of binding the nucleicacid binding protein binding element 1 and a second nucleic acid bindingprotein is capable of binding the nucleic acid binding protein bindingelement 2. In some embodiments, the nucleic acid binding protein bindingelement is a double-stranded nucleic acid binding protein bindingelement that binds a double-stranded nucleic acid binding protein.

In additional embodiments, a first repeat nucleic acid sequence of apair is connected with the second repeat nucleic acid sequence of thepair through hydrogen-bonded base pairs.

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H,and FIG. 7I, illustrate elements and examples of engineered concatenatednucleic acid scaffolds of the present invention.

Table 6 presents a series of indicators used consistently in FIG. 7A,FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H, and FIG.7I.

TABLE 6 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region an engineered NASC polynucleotideconcatenated element (corresponds- 700-717) (NASC-PC-CE) a firstconcatenate element 1 (NASC-PC-CE1-1) a nucleic acid binding proteinbinding element 1 700-701 corresponds to a linker element nucleic acidsequence 1-5 701-702 corresponds to a hairpin nucleic acid sequence 1-2702-703 corresponds to a linker element nucleic acid sequence 1-4703-704 corresponds to a hairpin nucleic acid sequence 1-1 704-705corresponds to a linker element nucleic acid sequence 1-3 705-706corresponds to a nexus element 1 706-707 corresponds to a linker elementnucleic acid sequence 1-2 a second concatenate element 1 (NASC-PC-CE1-2)a repeat element A1¹ 707-708 corresponds to a repeat nucleic acidsequence A1 707-728 corresponds to a linker element nucleic acidsequence A1-1 707-726 corresponds to a linker element nucleic acidsequence A1-4 726-727 corresponds to a repeat nucleic acid sequence A1-1727-728 corresponds to a bulge nucleic acid sequence A1-1 728-708corresponds to a linker element nucleic acid sequence A1-2 728-729corresponds to a repeat nucleic acid sequence A1-2 729-730 correspondsto a linker element nucleic acid sequence A1-3 730-708 corresponds to alinker nucleic sequence A1-4 that can comprise an effector proteinbinding site nucleic acid sequence A1 708-709 corresponds to a linkerelement nucleic acid sequence A1 a first concatenate element 2(NASC-PC-CE-1-2) a nucleic acid binding protein binding element 2(NASC-PC-CE2) 709-710 corresponds to a linker element nucleic acidsequence 2-5 709-710 corresponds to a linker element nucleic acidsequence 2-5 710-711 corresponds to a hairpin nucleic acid sequence 2-1711-712 corresponds to a linker element nucleic acid sequence 2-4712-713 corresponds to a linker element nucleic acid sequence 2-3713-714 corresponds to a nexus element 2 714-715 corresponds to a linkerelement nucleic acid sequence 2-2 a second concatenate element 2(NASC-PC-CE2-2) a repeat element A2 715-716 corresponds to a repeatnucleic acid sequence A2 715-731 corresponds to a linker element nucleicacid sequence A2-4 715-733 corresponds to a linker element nucleic acidsequence A2-1 731-732 corresponds to a repeat nucleic acid sequence A2-1732-733 corresponds to a bulge nucleic acid sequence A2-1 733-716corresponds to a linker element nucleic acid sequence A2-2 733-734corresponds to a repeat nucleic acid sequence A2-2 734-735 correspondsto a linker element nucleic acid A2-3 716-735 corresponds to a linkernucleic sequence A2-4 that can comprise an effector protein binding sitenucleic acid sequence A2 716-717 corresponds to linker element nucleicacid sequence A2 a third concatenate element 1 (NASC-PC-CE3-1) a repeatelement A1C² 718-719 corresponds to a repeat nucleic acid sequence A1C719-737 corresponds to a linker element nucleic acid sequence A1-1n719-736 corresponds to a repeat nucleic acid sequence A1-1C² 736-737corresponds to a bulge nucleic acid sequence A1-1n³ 737-718 correspondsto a linker element nucleic acid sequence A1-2n 737-748 corresponds to arepeat nucleic acid sequence A1-2C 738-748 corresponds to a linkerelement nucleic acid A1-3n 738-718 corresponds to a linker nucleicsequence A1-4n that can comprise an effector protein binding sitenucleic acid sequence A1C 719-720 corresponds to a linker elementnucleic acid sequence A1-4n a spacer element 1 720-721 corresponds anucleic acid target binding sequence 1 a third concatenate element 2(NASC-PC-CE3-2) a repeat element A2C 722-723 corresponds to a repeatnucleic acid sequence A2-1C2 723-740 corresponds to a linker elementnucleic acid sequence A2-1n 723-739 corresponds to a repeat nucleic acidsequence A2-1C 739-740 corresponds to a bulge nucleic acid sequenceA2-1n 722-740 corresponds to a linker element nucleic acid sequenceA2-2n 740-741 corresponds to a repeat nucleic acid sequence A2-2C741-749 corresponds to a a linker element nucleic acid A2-3n 749-722corresponds to a linker nucleic sequence A2-4 that can comprise aneffector protein binding site nucleic acid sequence A2C 723-724corresponds to a linker element nucleic acid sequence A2-4n a spacerelement 2 724-725 corresponds to a nucleic acid target binding sequence2 ¹= repeat element can include an effector protein binding site ²= “C”indicates a complementary sequence ³= “n” indicates an opposite strandsequence (e.g., A2-1/A2-1n)

Each of a first, a second and a third element can comprise additionalnucleic acid sequences, for example, 5′ of the element, 3′ of theelement, or both 5′ of the element and 3′ of the element.

FIG. 7A, 700-717, illustrates an example of a NASC-PC-CE that comprisesa first concatenate element 1 comprising a Class 2 Type II CRISPRbinding protein sequence (FIG. 7A, 700-706), a second concatenateelement 1 (FIG. 7A, 707-708) comprising a repeat nucleic acid sequenceA1, a first concatenate element 2 (FIG. 7A, 709-714) comprising a Class2 Type II CRISPR binding protein sequence (FIG. 7A, 710-714), a secondconcatenate element 2 (FIG. 7A, 715-717) comprising a repeat nucleicacid sequence A2 (FIG. 7A, 715-716), and a third concatenate element 1(NASC-PC-CE3-1; FIG. 7A, 718-721) comprising a repeat nucleic acidsequence A1C (FIG. 7A, 718-719) and a nucleic acid target bindingsequence 1 (FIG. 7A, 720-721), and a third concatenate element 2(NASC-PC-CE3-2; FIG. 7A, 722-725) comprising a repeat nucleic acidsequence A2C (FIG. 7A, 722-723) and a nucleic acid target bindingsequence 2 (FIG. 7A, 724-725). One or more of the repeat nucleic acidsequences is a Class II Type II CRISPR protein binding sequence (e.g., aCas9 protein binding sequence). Repeat nucleic acid sequence A1 isconnected to repeat nucleic acid sequence A1C. In one embodiment, repeatnucleic acid sequence A1 is connected through hydrogen-bonded base pairsto repeat nucleic acid sequence.

FIG. 7B illustrates an example of the formation of a scaffold throughassociation of the NASC-PC-CE (FIG. 7A, 700-717) with the thirdconcatenate element 1 (FIG. 7A, 718-721) through hydrogen base-pairbonding between repeat nucleic acid sequence A1 (FIG. 7A, 707-708) andrepeat nucleic acid sequence A1C (FIG. 7A, 718-719), and the associationof the NASC-PC-CE (FIG. 7A, 700-717) with the third concatenate element2 (FIG. 7A, 722-725) through hydrogen base-pair bonding between repeatnucleic acid sequence A2 (FIG. 7A, 715-716) and repeat nucleic acidsequence A2C (FIG. 7A, 722-723).

FIG. 7C illustrates a modification of a NASC-PC-CE wherein theNASC-PC-CE (FIG. 7A, 700-717) further comprises a repeat nucleic acidsequence A1-1 (FIG. 7C, 726-727), a bulge nucleic acid sequence A1-1(FIG. 7C, 727-728), a repeat nucleic acid sequence A1-2 (FIG. 7C,728-729) and a repeat nucleic acid sequence A2-1 (FIG. 7C, 731-732), abulge nucleic acid sequence A2-1 (FIG. 7C, 732-733), and a repeatnucleic acid sequence A2-2 (FIG. 7C, 733-734). The third concatenateelement 1 (FIG. 7C, 718-721) further comprises a repeat nucleic acidsequence A1-1C (FIG. 7C, 719-736), a bulge nucleic acid sequence A1-1(FIG. 7C, 736-737), and a repeat nucleic acid sequence A1-2C (FIG. 7C,737-748). The third concatenate element 2 (FIG. 7C, 722-725) furthercomprises a repeat nucleic acid sequence A2-1C (FIG. 7C, 723-739), abulge nucleic acid sequence A2-1 (FIG. 7C, 739-740), and a repeatnucleic acid sequence A2-2C (FIG. 7C, 740-741).

FIG. 7D illustrates an example of the formation of a scaffold throughassociation of the NASC-PC-CE (FIG. 7D, 700-717) with the thirdconcatenate element 1 (FIG. 7D, 721-718) through hydrogen base-pairbonding between: repeat nucleic acid sequence A1-1 (FIG. 7C, 726-727)and repeat nucleic acid sequence A1-1C (FIG. 7C, 719-736), and hydrogenbase-pair bonding between repeat nucleic acid sequence A1-2 (FIG. 7C,728-729) and repeat nucleic acid sequence A1-2C (FIG. 7C, 737-748);association of the engineered concatenated element 1 (FIG. 7D, 700-717)with the third concatenate element 2 (FIG. 7C, 722-725) through hydrogenbase-pair bonding between repeat nucleic acid sequence A2-1 (FIG. 7C,731-732) and repeat nucleic acid sequence A2-1C (FIG. 7C, 723-739), andthrough hydrogen base-pair bonding between repeat nucleic acid sequenceA2-2 (FIG. 7C, 733-734) and repeat nucleic acid sequence A2-2C (FIG. 7C,740-741).

FIG. 7E presents an example of a complex formed from four engineerednucleic acid sequences to make a scaffold comprising a circularNASC-PC-CE. In this figure, the NASC-PC-CE comprises two copies of theNASC-PC-CE shown in FIG. 7D, 700-717 that are joined 5′ end to 3′ end toform the circular NASC-PC-CE. In this figure, reference numbers relativeto FIG. 7D are shown to help illustrate the components of the circularconcatenated nucleic acid element.

FIG. 7F is an illustration of a modification to the example shown inFIG. 7D, wherein the NASC-PC-CE (FIG. 7F, 700-717) further comprises afirst concatenate element 3 (FIG. 7F, 717-744) covalently linked to the5′ end (FIG. 7F, 700-744). A second concatenate element 3 is associatedwith the first concatenate element 3 (FIG. 7F, 743-744).

FIG. 7G illustrates an example of a modification to the NASC-PC-CE (FIG.7F, 700-744) depicted in FIG. 7F, wherein the NASC-PC-CE comprises afourth concatenate element (FIG. 7G, 744-747) covalently linked to the5′ end (FIG. 7F, 700-747). In this figure, the region, FIG. 7G, 744-745,is illustrated as a white box to make the cross-over lines in FIG. 7Hand FIG. 7I more apparent. This region can also comprise a linkerelement nucleic acid sequence.

FIG. 7H, 700-747, illustrate an example of a NASC-PC-CE, wherein thesecond concatenate element 1 (FIG. 7H, 707-708) is associated with thethird concatenate element 1 (FIG. 7H, 744-747) through hydrogenbase-pair bonding.

FIG. 7I illustrates a modification to the example shown in FIG. 7H,wherein a third concatenated element I associates with the NASC-PC-CEthough hydrogen base-pair bonding to form element III (FIG. 7I, III) anda fourth concatenate element associates with the NASC-PC-CE thoughhydrogen base-pair bonding to form element IV (FIG. 7I, IV).

In other embodiments of the third aspect of the present invention, theNASC-PC-CE comprises split-nexus polynucleotides.

FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H,FIG. 8I, FIG. 8J, FIG. 8K, FIG. 8L, FIG. 8M, and FIG. 8N illustrateelements and examples of engineered concatenated split-nexus nucleicacid scaffolds of the present invention.

Table 7 presents a series of indicators used consistently in FIG. 8Athrough 8N.

TABLE 7 Numerical Indicators Used to Illustrate Regions of Complexes ofTwo or More Engineered Nucleic Acid Sequences for Forming a ScaffoldIndicator and Corresponding Region an engineered NASC split-nexuspolynucleotide concatenated element (corresponds to 800-835)(NASC-PC-SCE) a first concatenate element 1 (NASC-PC-SCE1-1) a nucleicacid binding protein binding element 1 800-801 corresponds to a nucleicacid binding protein binding nucleic acid 1 801-802 corresponds to asplit-nexus stem element nucleic acid sequence 1-1 a repeat element 1-1¹802-816 corresponds an auxiliary polynucleotide 1-1 802-815 correspondsto a repeat nucleic acid sequence 1-1 814-815 corresponds to adouble-stranded nucleic acid binding effector protein binding sitenucleic acid sequence 1 a second concatenate element 1 (NASC-PC-SCE1-2)a nucleic acid binding protein binding element 2 816-820 corresponds toa nucleic acid binding protein binding nucleic acid 2 820-821corresponds to a split-nexus stem element nucleic acid sequence 2-1 arepeat element 2-1 821-835 corresponds an auxiliary polynucleotide 2-1821-834 corresponds to a repeat nucleic acid sequence 2-1 833-834corresponds to a double-stranded nucleic acid binding effector proteinbinding site nucleic acid sequence 2 a second concatenate element 1(NASC-PC-SCE2-1) a repeat element 1 808-807 corresponds to a first stemelement nucleic acid sequence 1-1 808-810 corresponds to a lower stemelement nucleic acid sequence 1-1 810-811 corresponds to a bulge elementnucleic acid sequence 1-1 811-807 corresponds to an upper stem elementnucleic acid sequence 1-1 807-806 corresponds to a loop element nucleicacid sequence 1 806-805 corresponds to a first stem element nucleic acidsequence 1-2 806-812 corresponds to an upper stem element nucleic acidsequence 1-2 812-813 corresponds to a bulge element nucleic acidsequence 1-2 813-805 corresponds to a lower stem element nucleic acidsequence 1-2 805-804 corresponds to a connective nucleic acid sequence 1[805-806/808-807 corresponds a first stem element 1] [811-807/806-812corresponds to an upper stem element 1] [810-811/812-813 corresponds toa bulge element 1] [808-810/813-805 corresponds to a lower stem element1] 804-803 corresponds to a split-nexus stem element nucleic acidsequence 1-2 [803-804/801-802 corresponds to a nexus element 1] 803-817corresponds to an auxiliary polynucleotide 1-2 803-818 corresponds to arepeat nucleic acid sequence 1-2 (in some embodiments complementary tothe repeat nucleic acid sequence 1-1) 818-819 corresponds to adouble-stranded nucleic acid binding effector protein binding sitenucleic acid sequence 1C² [the site 814-815/818-819 corresponds to adouble-stranded nucleic acid binding effector protein binding site 1] aspacer element 1 808-809 corresponds to a nucleic acid target bindingsequence 1 a second concatenate element 1 (NASC-PC-SCE2--2) a repeatelement 2 827-826 corresponds to a first stem element nucleic acidsequence 2-1 832-827 corresponds to a lower stem element nucleic acidsequence 2-1 831-832 corresponds to a bulge element nucleic acidsequence 2-1 831-826 corresponds to an upper stem element nucleic acidsequence 2-1 825-826 corresponds to a loop element nucleic acid sequence2 824-825 corresponds to a first stem element nucleic acid sequence 2-2825-830 corresponds to an upper stem element nucleic acid sequence 2-2829-830 corresponds to a bulge element nucleic acid sequence 2-2 829-824corresponds to a lower stem element nucleic acid sequence 2-2 823-824corresponds to a connective nucleic acid sequence 2 [826-827/824-825corresponds a first stem element 2] [826-831/825-830 corresponds to anupper stem element 2] [831-832/829-830 corresponds to a bulge element 2][827-832/824-829 corresponds to a lower stem element 2] 822-823corresponds to a split-nexus stem element nucleic acid sequence 2-2[822-823/820-821 corresponds to a nexus element 2] 822-836 correspondsto an auxiliary polynucleotide 2-2 822-837 corresponds to a repeatnucleic acid sequence 2-2 (in some embodiments complementary to therepeat nucleic acid sequence 2-1) 837-838 corresponds to adouble-stranded nucleic acid binding effector protein binding sitenucleic acid sequence 2C [the site 837-838/833-834 corresponds to adouble-stranded nucleic acid binding effector protein binding site 2] aspacer element 2 827-828 corresponds to a nucleic acid target bindingsequence 2 ¹= a repeat element can include an effector protein bindingsite ²= “C” indicates a complementary sequence

FIG. 8A illustrates an example of split-nexus Cas9-associatedpolynucleotides. FIG. 2B presents an example of a Cas9-associatedsingle-guide polynucleotide. The split-nexus Cas9-associatedpolynucleotides of FIG. 8A are generated by splitting the polynucleotidebackbone within the nexus element (FIG. 2B, 206) of a Cas9-associatedsingle-guide polynucleotide. FIG. 8A shows the two resulting split-nexuspolynucleotides when not associated through hydrogen bond interactions.FIG. 8B presents a view of the split-nexus polynucleotides whenassociated through hydrogen bond interactions. The region of thehydrogen bond interactions is illustrated by a broken-dash box in FIG.8B.

FIG. 8C illustrates another example of split-nexus Cas9-associatedpolynucleotides. FIG. 2A presents an example of a Cas9-associatedsingle-guide polynucleotide. The split-nexus Cas9-associatedpolynucleotides of FIG. 8C are generated by splitting the polynucleotidebackbone within the nexus element (FIG. 2A, 206) of a Cas9-associatedsingle-guide polynucleotide. FIG. 8C shows the two resulting split-nexuspolynucleotides when not associated through hydrogen bond interactions.FIG. 8D presents a view of the split-nexus polynucleotides whenassociated through hydrogen bond interactions. The region of thehydrogen bond interactions is illustrated by a broken-dash box in FIG.8D.

FIG. 8E illustrates the addition of an auxiliary polynucleotide to thesplit-nexus Cas9-associated polynucleotide illustrated in FIG. 8A. Inthis figure, the 5′ end of a first auxiliary polynucleotide (FIG. 8E,803-817) is covalently attached to the 3′ end of one half of thesplit-nexus element (FIG. 8E, 803) and the 3′ end of a second auxiliarypolynucleotide (FIG. 8E, 802-816) is covalently attached to the 5′ endof the other half of the split-nexus element (FIG. 8E, 802). In someembodiments, only one auxiliary polynucleotide is included. In otherembodiments, two auxiliary polynucleotides of the same or differentlengths are included. FIG. 8E shows the two split-nexus polynucleotideswhen not associated through hydrogen bond interactions.

FIG. 8F presents a view of the split-nexus polynucleotides whenassociated through hydrogen bond interactions. The region of thehydrogen bond interactions is illustrated by the broken-dash box at FIG.8D, 803-804/801-802. An auxiliary polynucleotide can comprise additionalelements such as effector protein binding sequences, for example, adouble-stranded nucleic acid binding protein binding site can be createdby the association of two auxiliary polynucleotides through hydrogenbond interactions (e.g., such region of the hydrogen bond interactionsis illustrated by the broken-dash box at FIG. 8D, 818-819-804/814-815).

FIG. 8G illustrates another example of the addition of an auxiliarypolynucleotide to the split-nexus Cas9-associated polynucleotideillustrated in FIG. 8C. In this figure, the 5′ end of a first auxiliarypolynucleotide (FIG. 8G, 803-817) is covalently attached to the 3′ endof one half of the split-nexus element (FIG. 8E, 803) and the 3′ end ofa second auxiliary polynucleotide (FIG. 8G, 802-816) is covalentlyattached to the 5′ end of the other half of the split-nexus element(FIG. 8G, 802). In some embodiments, only one auxiliary polynucleotideis included. In other embodiments, two auxiliary polynucleotides of thesame or different lengths are included. FIG. 8G shows the twosplit-nexus polynucleotides when not associated through hydrogen bondinteractions. FIG. 8H presents a view of the split-nexus polynucleotideswhen associated through hydrogen bond interactions. The region of thehydrogen bond interactions is illustrated by the broken-dash box at FIG.8H, 803-804/801-802. An auxiliary polynucleotide can comprise additionalelements such as effector protein binding sequences, for example, adouble-stranded nucleic acid binding protein binding site can be createdby the association of two auxiliary polynucleotides through hydrogenbond interactions (e.g., such region of the hydrogen bond interactionsis illustrated by the broken-dash box at FIG. 8H, 818-819-804/814-815).

In one embodiment, the third aspect of the present invention is directedto an engineered NASC split-nexus polynucleotide concatenated element(NASC-PC-SCE) polynucleotide composition comprising, in a 3′ to 5′direction, a first concatenate element 1 (NASC-PC-SCE1-1) comprising anucleic acid binding protein binding element 1, a split-nexus stemelement nucleic acid sequence 1-1, and a repeat element comprising arepeat nucleic acid sequence 1-1, and a second concatenate element 1(NASC-PC-SCE1-2) comprising a nucleic acid binding protein bindingelement 2, a split-nexus stem element nucleic acid sequence 2-1, and arepeat element comprising a repeat nucleic acid sequence 2-1. TheNASC-PC-SCE1-1 and the NASC-PC-SCE1-2 are connected to form theNASC-PC-SCE. A second concatenate element 1 (NASC-PC-SCE2-1) comprises arepeat element 1 comprising, in a 3′ to 5′ direction, a repeat nucleicacid sequence 1-2, a split-nexus stem element nucleic acid sequence 1-2,and a first stem element, and a spacer element 1 comprising a nucleicacid target binding sequence 1. A second concatenate element 2(NASC-PC-SCE2-2) comprises a repeat element 2 comprising, in a 3′ to 5′direction, a repeat nucleic acid sequence 2-2, a split-nexus stemelement nucleic acid sequence 2-2, and a first stem element, and aspacer element 2 comprising a nucleic acid target binding sequence 2.

The repeat element 1-1 is connected to the repeat element 1-2, and therepeat element 2-1 is connected to the repeat element 2-2 to form theNASC-PC-SCE, and the NASC-PC-SCE is capable of binding two nucleic acidbinding proteins. In some embodiments, the nucleic acid binding proteinbinding element is a double-stranded nucleic acid binding proteinbinding element that binds a double-stranded nucleic acid bindingprotein. In additional embodiments, a first repeat nucleic acid sequenceof a pair is connected with the second repeat nucleic acid sequence ofthe pair through hydrogen-bonded base pairs.

FIG. 8I presents an example of a NASC-PC-SCE comprising two copies ofthe split-nexus polynucleotide shown in FIG. 8B, 800-802 forming ascaffold. In this figure, the NASC-PC-SCE is a first split-nexuspolynucleotide (FIG. 8I, 800-802) covalently attached to a secondsplit-nexus polynucleotide (FIG. 8I, 816-821) through an auxiliarypolynucleotide (FIG. 8I, 802-816). Each first half of a split-nexuselement (FIG. 8I, 801-802 and 820-821) is connected with thecomplementary second half of its split-nexus element (FIG. 8I, 803-804,and 822-823, respectively), for example, through hydrogen-bonded basepairs.

FIG. 8J presents an example of a NASC-PC-SCE comprising two copies ofthe split-nexus polynucleotide shown in FIG. 8D, 800-802 forming ascaffold. In this figure, the NASC-PC-SCE is a first split-nexuspolynucleotide (FIG. 8J, 800-802) covalently attached to a secondsplit-nexus polynucleotide (FIG. 8J, 816-821) through an auxiliarypolynucleotide (FIG. 8J, 802-816). Each first half of a split-nexuselement (FIG. 8J, 801-802 and 820-821) is connected with thecomplementary second half of its split-nexus element (FIG. 8J, 803-804,and 822-823, respectively), for example, through hydrogen-bonded basepairs.

FIG. 8K presents an example of a NASC-PC-SCE comprising two copies ofthe split-nexus polynucleotide shown in FIG. 8F, 800-816, eachcomprising an auxiliary sequence, forming a scaffold. In this figure,the NASC-PC-SCE is a first split-nexus polynucleotide (FIG. 8K, 800-816)covalently attached to a second split-nexus polynucleotide (FIG. 8J,816-835) through an auxiliary polynucleotide (FIG. 8J, 802-816). Eachfirst half of a split-nexus element (FIG. 8K, 801-802 and 820-821) isconnected with the complementary second half of its split-nexus element(FIG. 8K, 803-804, and 822-823, respectively) and is also connected bythe auxiliary sequences (FIG. 8K, 802-815 and 803-818; FIG. 8K, 821-834and 822-836). The connections are made, for example, throughhydrogen-bonded base pairs.

FIG. 8L presents an example of a NASC-PC-SCE comprising two copies ofthe split-nexus polynucleotide shown in FIG. 8H, 800-816, eachcomprising an auxiliary sequence, forming a scaffold. In this figure,the NASC-PC-SCE is a first split-nexus polynucleotide (FIG. 8H, 800-816)covalently attached to a second split-nexus polynucleotide (FIG. 8H,816-835) through an auxiliary polynucleotide (FIG. 8H, 802-816). Eachfirst half of a split-nexus element (FIG. 8H, 801-802 and 820-821) isconnected with the complementary second half of its split-nexus element(FIG. 8H, 803-804, and 822-823, respectively) and is also connected bythe auxiliary sequences (FIG. 8H, 802-815 and 803-818; FIG. 8K, 821-834and 822-836). The connections are made, for example, throughhydrogen-bonded base pairs. The two components of the NASC-PC-SCE areindicated in this figure as I and II.

FIG. 8M presents an example of a NASC-PC-SCE comprising elements I andII as shown in FIG. 8L. NASC-PC-SCE comprises a circular NASC-PC-SCE. Inthis figure, two sets of elements I and II, shown in FIG. 8L, 800-835,are joined 5′ end to 3′ end to form the circular NASC-PC-SCE. In thisfigure, reference numbers relative to FIG. 8L are shown to helpillustrate the components of the circular NASC-PC-SCE.

FIG. 8N presents an example of a NASC-PC-SCE comprising elements I andII as shown in FIG. 8L, with the exception that the first stem elementnucleic acid sequences are not joined by loop element nucleic acidsequences (see, e.g., FIG. 8L, 806-807, 825-826). NASC-PC-SCE comprisesa circular NASC-PC-SCE.

In other embodiments, a first repeat nucleic acid sequence of a pairfurther comprises first affinity tag and the second repeat nucleic acidsequence of the pair further comprises a second affinity tag, and thefirst affinity tag is connected with the second affinity tag. Forexample, the first repeat nucleic acid sequence further comprises aneffector protein binding site nucleic acid sequence 1 and the secondrepeat nucleic acid sequence further comprises an effector proteinbinding site nucleic acid sequence 2. The effector protein binding sitenucleic acid sequence 1 is connected by hydrogen-bonded base pairs tothe effector protein binding site nucleic acid sequence 2 to form aneffector protein binding site 1. One example of an effector binding siteis a Csy4 protein binding site.

In a fourth aspect of the present invention, a NASC polynucleotidecomposition comprises a combination of nucleic acid binding proteinbinding elements for two or more different nucleic acid bindingproteins. In some embodiments, one or more of the nucleic acid bindingprotein binding elements is a double-stranded nucleic acid bindingprotein binding element that binds a double-stranded nucleic acidbinding protein (e.g., a Class 2 CRISPR-Cas protein). Embodiments of thefourth aspect of the present invention include a first NATNA covalentlyconnected to a second NATNA to form a NASC polynucleotide composition.In some embodiments, a first NATNA covalently is connected to a secondNATNA to form a NASC-PC component and two or more NASC-PC components areconnected either covalently or non-covalently to form a NASCpolynucleotide composition. The NASC polynucleotide composition iscapable of binding at least two nucleic acid binding proteins.Non-covalent connections include connecting the NASC-PC componentsthrough hydrogen-bonded base pairs.

FIG. 9A presents an example of two NATNAs joined to form a NASCpolynucleotide composition comprising (i) a copy of the engineerednucleic acid sequence shown in FIG. 5D, I, 500-507 (FIG. 9A, I), and(ii) a copy of an engineered nucleic acid sequence corresponding to thesingle-guide polynucleotide shown in FIG. 2A further comprising a linkerelement nucleic acid sequence covalently attached to the 3′ end of thesingle-guide polynucleotide (FIG. 9A, II), wherein the linker elementnucleic acid sequence is covalently attached to the 5′ end of theengineered nucleic acid sequence shown in FIG. 5D, I, 500-507, forming ascaffold.

FIG. 9B presents an example of a complex of two sets of the componentsshown in FIG. 9A. In this figure, reference numbers relative to FIG. 9A,I and II, are shown to help illustrate the components. Furthermore, FIG.9A, III, is provided to facilitate comparison of the core structure ofthe complex of FIG. 9A to the complex presented in FIG. 5D.

FIG. 10 presents a complex of a number of different engineered nucleicacid sequences forming a scaffold. In this figure, reference numbers areprovided to help illustrate the components of the scaffold: FIG. 10, I,compare to FIG. 7F; FIG. 10, II, compare to FIG. 6H; FIG. 10, III,compare to FIG. 8L; and FIG. 10, IV, compare to FIG. 9A, wherein I andII are connected through hydrogen-bonded base pairs instead of acovalent connection; and FIG. 10, V compare to FIG. 5H.

The types of connections between one or more polynucleotide componentsof a NASC polynucleotide composition include, for example, covalentlinkages and non-covalent linkages.

One example of a non-covalent linkage is hydrogen bonding. Types ofhydrogen bonds are discussed above. Embodiments of the present inventioninclude, but are not limited to, the following types of hydrogen bondsin pairs of hydrogen-bonded nucleotides: W—C hydrogen bonding, reverseW—C hydrogen bonding, Hoogsteen hydrogen bonding, reverse Hoogsteenhydrogen bonding, wobble hydrogen bonding, reverse wobble hydrogenbonding, or combinations thereof.

NASC polynucleotide components are typically designed such that pairedrepeat elements intended to connect with each other, particularly if theconnection is through hydrogen-bonded base pairs, and only formconnections (e.g., hydrogen bonds) between the paired repeat elements.Formation of internal structures within each repeat element thatinterfere with the two repeat elements connecting typically avoided.Furthermore, connections (e.g., formation of hydrogen-bonded base pairs)between the repeat elements and other regions of component NASCpolynucleotides are also avoided.

In addition to covalent linkages and non-covalent linkages, other typesof connections between one or more polynucleotide components of a NASCpolynucleotide composition can be used including, but not limited to,ligand/ligand binding moiety pairings, and/or cross-linking.Ligand/ligand binding moiety pairings include, but are not limited to, aselected nucleic acid sequence and a corresponding aptamer; and anucleic acid secondary structure/a small molecule, ion, or protein thatbinds to the nucleic acid secondary structure. Typically, a firstpolynucleotide component of a NASC polynucleotide composition is adaptedto comprise a ligand (e.g., the first polynucleotide component of a NASCpolynucleotide composition comprises at its 3′ end a selected nucleicacid sequence) and a second polynucleotide component of the NASCpolynucleotide composition is adapted to comprise a ligand bindingmoiety (e.g., the second polynucleotide component of the NASCpolynucleotide composition comprises an aptamer at its 5′ end that bindsthe selected nucleic acid sequence).

Cross-linking agents useful to form connections between one or morepolynucleotide components of a NASC polynucleotide composition include,but are not limited to, alkylating agents (e.g.,1,3-bis(2-chloroethyl)-1-nitrosourea) and nitrogen mustard); cisplatin(cis-diamminedichloroplatinum(II)) and its derivatives); ionizingradiation; nitrous acid; reactive chemicals (e.g., malondialdehyde);psoralens (activated in the presence of UV); and aldehydes (e.g.,acrolein and crotonaldehyde).

In some embodiments of the present invention, affinity tags areintroduced into two or more polynucleotide components of a NASCpolynucleotide composition. For example, a nucleic acid sequence withinone polynucleotide component of a NASC polynucleotide composition can bemodified to comprise an affinity sequence. Nucleic acid binding effectorproteins and their corresponding effector protein binding sequences areexamples of affinity tags. An affinity tag can be introduced into afirst polynucleotide components of a NASC polynucleotide composition. Anaffinity tag can be an affinity sequences such as MS2 binding sequence,U1A binding sequence, stem-loop sequence (e.g., a Csy4 protein bindingsequence, or Cas6 protein binding sequence), eIF4A binding sequence,Transcription Activator-Like Effector (TALE) binding sequence (see,e.g., Valton, J., et al., Journal of Biological Chemistry287(46):38427-38432 (2012)), or zinc finger domain binding sequence(see, e.g., Font, J., et al., Methods Molecular Biology 649:479-491(2010); Isalan, M., et al., Nature Biotechnology 19(7):656-660 (2001)).A second polynucleotide component of the NASC polynucleotide compositioncan be modified to comprise a corresponding affinity tag: an MS2 codingsequence, U1A coding sequence, stem-loop binding protein coding sequence(e.g., an enzymatically (endoribonuclease) inactive Csy4 protein thatbinds the Csy4 protein sequence), eIF4A coding sequence, TALE codingsequence, or a zinc finger domain coding sequence, respectively.Typically, enzymatically inactive nucleic acid binding proteins thatretain sequence specific nucleic acid binding are used (e.g., anendoribonuclease-inactive Csy4 protein (dCsy4)); however, in someembodiments enzymatically active nucleic acid binding proteins ornucleic acid proteins with altered enzymatic activity are used. Whenmore than two polynucleotide components of a NASC polynucleotidecomposition are modified with an affinity sequence, in preferredembodiments, the two affinity sequences typically are not the same;thus, there are two different affinity sequences associated with the Casprotein.

Example 1 describes production of exemplary components of engineeredNASC polynucleotide compositions. Example 1 describes in silico designof NASC polynucleotide components corresponding to a number ofembodiments of the NASC polynucleotide compositions described herein.Table 9 sets forth a correlation between NASC polynucleotide componentsand structures illustrated in the figures.

Example 2 describes production of NASC polynucleotide components of thepresent invention. The NASC polynucleotide components described in thisExample were used in in vitro Cas cleavage assays to evaluate cleavagepercentages of nucleic acid target sequences by the NASC polynucleotidecompositions. Example 5 describes performance of in vitro Casprotein-mediated cleavage assays. Example 3 and Example 4 describemethods that can be used for production of double-stranded DNA targetsequences for use in in vitro Cas cleavage assays.

Example 6 presents a deep sequencing analysis for detection of targetmodifications in eukaryotic cells using NASC polynucleotidecompositions/first nucleic acid binding protein/second nucleic acidbinding protein compositions (comprising, for example, Class 2CRISPR-Cas proteins) of the present invention.

Example 9 presents an alternative analysis, the T7E1 assay, fordetection of target modifications in eukaryotic cells using NASCpolynucleotide composition/first nucleic acid binding protein/secondnucleic acid binding protein compositions (comprising, for example,Class 2 CRISPR-Cas proteins).

Example 7 describes identification and screening of Class 2 crRNAs thatcan be engineered to make NASC polynucleotide components of the presentinvention.

Example 8 describes identification and screening of Class 2 tracrRNAsthat can be used to engineer NASC polynucleotide components.

Example 10 describes the generation and testing of various modificationsof Class 2 Type V guide crRNAs and their suitability for use inconstructing NASC polynucleotide components.

Example 11 describes the generation and testing of various modificationsof Class 2 Type II guide RNAs and their suitability for use inconstructing NASC polynucleotide components.

Example 12 describes the use of NASC polynucleotide compositions tomodify nucleic acid target sequences present in human gDNA and measurethe level of cleavage activity and specificity of cleavage at thosesites. Measurement of the level of cleavage percentage and/or cleavagespecificity at a particular site can provide options to identify thenucleic acid target sequences having a desired cleavage percentageand/or specificity.

In a fifth aspect, the present invention is directed to nucleicacid/protein compositions comprising a NASC polynucleotide compositioncomplexed with a first nucleic acid binding protein and a second nucleicacid binding protein. The first nucleic acid binding protein cancomprise one or more nuclease activities, and the second nucleic acidbinding protein can comprise one or more nuclease activities. In someembodiments, the first nucleic acid binding protein is catalyticallyinactive for one or more of the nuclease activities, the second nucleicacid binding protein is catalytically inactive for one or more of thenuclease activities, or both the first nucleic acid binding protein iscatalytically inactive for one or more of the nuclease activities andthe second nucleic acid binding protein is catalytically inactive forone or more of the nuclease activities. In other embodiments of the NASCpolynucleotide composition/first nucleic acid binding protein/secondnucleic acid binding protein complexes, either the first nucleic acidbinding protein or the second nucleic acid binding protein iscatalytically inactive, and the complexes can further be connected witha donor polynucleotide via the catalytically inactive protein. Inpreferred embodiments, the first nucleic acid binding protein and thesecond nucleic acid binding protein are Class 2 CRISPR-Cas proteins(e.g., a Cas9 protein, a Cpf1 protein, or a Cas9 protein and a Cpf1protein).

In some embodiments of the NASC polynucleotide composition/first nucleicacid binding protein/second nucleic acid binding protein composition,either the Cas9 protein or the Cpf1 protein is catalytically inactive(dCas9 or dCpf1) and the NASC polynucleotide composition/first nucleicacid binding protein/second nucleic acid binding protein compositionfurther comprises a donor polynucleotide wherein the donorpolynucleotide comprises a nucleotide sequence complementary to the Cpf1spacer element, or the regions adjacent to the Cpf1 spacer element, or anucleotide sequence complementary to the spacer element, or the regionsadjacent to the Cas9 spacer element. The donor polynucleotide is capableof associating with the spacer element, or the regions adjacent to thespacer element, through hydrogen bonding between the donorpolynucleotide nucleotide sequence complementary to the spacer element,or the sequence adjacent to the spacer element.

Mutations of the Cas9 protein that are enzymatically inactive forRuvC-1-related nuclease activity, HNH-related nuclease activity, andboth RuvC-1-related nuclease activity and HNH-related nuclease activityare known in the art. Mutations of the Cpf1 protein that areenzymatically inactive are known in the art (see, e.g., Yamano, T., etal., Cell 165(4):949-962 (2016)); Zetsche, B., et al., Cell 163:1-13(2015)).

Across CRISPR systems, “guide biogenesis” (also referred to as “guideprocessing”) involves endonuclease or exonuclease truncation of theguide RNA sequence following transcription of the CRISPR array.Enzymatic processing of the guide RNA can be carried out by RNasesencoded by the Cas operon (e.g., Cas6 of Class 1 Type I-E systems) or byendogenous RNases (e.g., RNase III of Class 2 Type II-A systems).

In Class 2 Type V systems, guide biogenesis is performed by the Cpf1protein nuclease. The Cpf1 protein is also responsible forsequence-specific double-stranded DNA target cleavage.

In the Type V system, cleavage of the pre-crRNA occurs in an upstreamregion (e.g., in a 5′ direction) from the pseudo-knot secondarystructure (see, e.g., FIG. 3A, 303) and results in the generation of aguide Cpf1 crRNA. In some embodiments of the present invention,preventing the Cpf1 protein from cleaving 5′ of the guide crRNA stemelement is useful, for example, to prevent separation of a NASCpolynucleotide composition/Cas9 protein/Cpf1 protein complex byCpf1-mediated cleavage. It has been demonstrated that the sequence ofType V pre-crRNA can be modified to prevent guide RNA processing by theType V CRISPR Cpf1 protein (see Fonfara, I., et al., Nature532(7600):517-521 (2016)).

One method to prevent Cpf1 cleavage of sequences 5′ of the guide crRNAstem element is by modification (e.g., base mutations, insertions,deletions, or chemical modifications) of the bases in the regionupstream of the pseudo-knot or within the pseudo-knot of the pre-crRNAto prevent the processing of the pre-crRNA by the Cpf1 protein. Toevaluate the effect of such modifications on guide processing, themodified pre-crRNA can be incubated in the presence of a cognate Cpf1protein for a period of time in a suitable buffer. The mixture can betreated with Proteinase K (Denville Scientific, South Plainfield, N.J.)to remove the protein and the mixture can be analyzed by polyacrylamidegel electrophoresis to evaluate whether cleavage of the modifiedpre-crRNA occurs. A pre-crRNA not incubated in the presence of a cognateCpf1 protein can serve as a positive control (i.e., a control for theabsence of guide processing). If no single modification in the pre-crRNAis sufficient to ablate guide processing, then combinations ofmodifications exhibiting reduced processing of the pre-crRNA can becombined into a pre-crRNA design and re-tested for the absence of guideprocessing activity. Modifications of pre-crRNA that result in theinability of the modified pre-crRNA to be processed can be furtherevaluated for the ability of the Cpf1-pre-crRNA/Cpf1 protein complex tomaintain sequence-specific binding and/or cleavage of a DNA targetnucleic acid comprising the pre-crRNA spacer element.

A second method to prevent Cpf1 cleavage of sequences 5′ of the guidecrRNA stem element is by modification of the Cpf1 protein. In thismethod, the amino acid residues of the Cpf1 protein are modified toperturb guide processing. X-ray crystallography of guide crRNA/Cpf1protein complexes has shown that the pseudo-knot is bound by theinterface of two protein domains designated the wedge domain (WED) andthe RuvC domain (see Yamano, T., et al., Cell 165(4):949-962 (2016).Amino acid residues of Cpf1 proximal to the region binding the 5′ end ofthe guide crRNA and/or the pseudo-knot structure are likely to beinvolved in endonuclease catalysis of pre-crRNAs. Mutagenesisstrategies, such as alanine screening (see, e.g., Lefèvre, F., et al.,Nucleic Acids Research 25(2):447-448 (1997); Lee, et al., MolecularPharmacology 50(1):140-148 (1996)) can be used to modify regions withinthe WED and RuvC domain, or other domains within the Cpf1 protein, toidentify residues in the protein responsible for guide crRNA processing.In this method, Cpf1 proteins comprising alanine mutations can beexpressed and incubated with a cognate pre-crRNA in a suitable buffer.After incubation, Proteinase K can be added to the reaction mix toremove the Cpf1 protein and the reaction mix can be analyzed bypolyacrylamide gel electrophoresis to evaluate whether cleavage of themodified pre-crRNA occurred. A pre-crRNA not incubated in the presenceof a cognate Cpf1 protein can serve as a positive control (i.e., acontrol for the absence of guide processing). If no single mutation inthe Cpf1 protein is sufficient to ablate guide processing, thencombinations of mutations exhibiting reduced processing of the pre-crRNAcan be combined into a single Cpf1 protein construct and re-tested forthe absence of guide processing activity. Candidate mutations orcombinations of mutations in the Cpf1 protein can be further evaluatedfor the ability of the Cpf1-pre-crRNA complex to maintainsequence-specific binding and/or cleavage of a DNA target nucleic acidcomprising the pre-crRNA spacer element.

In a sixth aspect, the present invention relates to nucleic acidsequences encoding one or more polynucleotide components of a NASCpolynucleotide composition, as well as expression cassettes, vectors,and recombinant cells comprising nucleic acid sequences encoding one ormore polynucleotide components of a NASC polynucleotide composition. Insome embodiments, such expression cassettes, vectors, and recombinantcells further comprise sequences encoding one or more nucleic acidbinding proteins (e.g., Class 2 CRISPR-Cas proteins) with which the NASCpolynucleotide composition is capable of forming a complex.

A further embodiment of the present invention relates to vectors,including expression vectors, comprising one or more nucleic acidsequences encoding one or more polynucleotide components of a NASCpolynucleotide composition, and optionally one or more nucleic acidsequences encoding nucleic acid binding proteins (e.g., Class 2CRISPR-Cas proteins) capable of forming a complex with the NASCpolynucleotide composition. Vectors can also include sequences encodingselectable or screenable markers. Furthermore, nuclear targetingsequences can also be added, for example, to Cas9 protein and Cpf1protein coding sequences. Vectors can also include polynucleotidesencoding protein tags (e.g., poly-His tags, hemagglutinin tags,fluorescent protein tags, bioluminescent tags). The coding sequences forsuch protein tags can be fused to, for example, one or more nucleic acidsequences encoding a Cas9 protein and/or a Cpf1 protein.

General methods for construction of expression vectors are known in theart; furthermore, expression vectors for host cells are commerciallyavailable. There are several commercial software products designed tofacilitate selection of appropriate vectors and construction thereof,such as insect cell vectors for insect cell transformation and geneexpression in insect cells, bacterial plasmids for bacterialtransformation and gene expression in bacterial cells, yeast plasmidsfor cell transformation and gene expression in yeast and other fungi,mammalian vectors for mammalian cell transformation and gene expressionin mammalian cells or mammals, and viral vectors (including lentivirus,retrovirus, adenovirus, herpes simplex virus I or II, parvovirus,reticuloendotheliosis virus, and adeno-associated virus (AAV) vectors)for cell transformation and gene expression and methods to easily allowcloning of such polynucleotides. Illustrative plant transformationvectors include those derived from a Ti plasmid of Agrobacteriumtumefaciens (Lee, L. Y., et al., Plant Physiology 146(2): 325-332(2008)). Also useful and known in the art are Agrobacterium rhizogenesplasmids. For example, SNAPGENE™ (GSL Biotech LLC, Chicago, Ill.;snapgene.com/resources/plasmid_files/your_time_is_valuable/) provides anextensive list of vectors, individual vector sequences, and vector maps,as well as commercial sources for many of the vectors.

Lentiviral vectors are examples of vectors useful for introduction intomammalian cells of one or more nucleic acid sequences encoding one ormore polynucleotide components of a NASC polynucleotide composition, andoptionally one or more nucleic acid sequences encoding one or morenucleic acid binding proteins (e.g., Class 2 CRISPR-Cas proteins) withwhich the NASC polynucleotide composition is capable of forming acomplex. Lentivirus is a member of the Retroviridae family and is asingle-stranded RNA virus, which can infect both dividing andnon-dividing cells as well as provide stable expression throughintegration into the genome. To increase the safety of lentivirus,components necessary to produce a viral vector are split across multipleplasmids. Transfer vectors are typically replication incompetent and mayadditionally contain a deletion in the 3′LTR, which renders the virusself-inactivating after integration. Packaging and envelope plasmids aretypically used in combination with a transfer vector. For example, apackaging plasmid can encode combinations of the Gag, Pol, Rev, and Tatgenes. A transfer plasmid can comprise viral LTRs and the psi packagingsignal. The envelope plasmid comprises an envelope protein (usuallyvesicular stomatitis virus glycoprotein, VSV-GP, because of its wideinfectivity range).

Lentiviral vectors based on human immunodeficiency virus type-1 (HIV-1)have additional accessory proteins that facilitate integration in theabsence of cell division. HIV-1 vectors have been designed to address anumber of safety concerns. These include separate expression of theviral genes in trans to prevent recombination events leading to thegeneration of replication-competent viruses. Furthermore, thedevelopment of self-inactivating vectors reduces the potential fortransactivation of neighboring genes and allows the incorporation ofregulatory elements to target gene expression to particular cell types(see, e.g., Cooray, S., et al., Methods in Enzymology 507:29-57 (2012)).

Transformed host cells (or recombinant cells) are cells or the progenyof cells that have been transformed or transfected, using recombinantDNA techniques, with one or more nucleic acid sequences encoding one ormore polynucleotide components of a NASC polynucleotide composition, andoptionally one or more nucleic acid sequences encoding one or morenucleic acid binding proteins (e.g., Class 2 CRISPR-Cas proteins) withwhich the NASC polynucleotide composition is capable of forming acomplex. Methods of introducing polynucleotides (e.g., an expressionvector) into host cells are known in the art and are typically selectedbased on the kind of host cell. Such methods include, for example, viralor bacteriophage infection, transfection, conjugation, electroporation,calcium phosphate precipitation, polyethyleneimine-mediatedtransfection, DEAE-dextran mediated transfection, protoplast fusion,lipofection, liposome-mediated transfection, ballistic gene transfertechnology (e.g., using a gene gun or a biolistic particle deliverysystem), direct microinjection, and nanoparticle-mediated delivery.

As an alternative to expressing one or more nucleic acid sequencesencoding one or more polynucleotide components of a NASC polynucleotidecomposition, and optionally one or more nucleic acid sequences encodingone or more nucleic acid binding proteins (e.g., Class 2 CRISPR-Casproteins) with which the NASC polynucleotide composition is capable offorming a complex, a NASC polynucleotide composition and/or the one ormore nucleic acid binding proteins (e.g., Class 2 CRISPR-Cas proteins)can be directly introduced into a cell, for example. Alternatively, oneor more components can be expressed by a cell and the other component(s)directly introduced. Methods to introduce the components into a cellinclude electroporation, lipofection, and ballistic gene transfertechnology.

A variety of host cells are disclosed herein that can be used to producerecombinant cells by introduction of one or more nucleic acid sequencesencoding one or more polynucleotide components of a NASC polynucleotidecomposition, and optionally one or more nucleic acid sequences encodingone or more nucleic acid binding proteins (e.g., Class 2 CRISPR-Casproteins) with which the NASC polynucleotide composition is capable offorming a complex. Such host cells include, but are not limited to aplant cell, a yeast cell, a bacterial cell, an insect cell, an algalcell, or a mammalian cell.

Methods of introducing polynucleotides (e.g., an expression vector) intohost cells to produce recombinant cells are known in the art and aretypically selected based on the kind of host cell. Such methods include,for example, viral or bacteriophage infection, transfection,conjugation, electroporation, calcium phosphate precipitation,polyethyleneimine-mediated transfection, DEAE-dextran mediatedtransfection, protoplast fusion, lipofection, liposome-mediatedtransfection, ballistic gene transfer technology, direct microinjection,and nanoparticle-mediated delivery. For ease of discussion,“transfection” is used below to refer to any method of introducingpolynucleotides into a host cell.

Preferred methods for introducing polynucleotides plant cells includemicroprojectile bombardment and Agrobacterium-mediated transformation.Alternatively, other non-Agrobacterium species (e.g., Rhizobium) andother prokaryotic cells that are able to infect plant cells andintroduce heterologous polynucleotides into the genome of the infectedplant cell can be used. Other methods include electroporation,liposome-mediated transfection, transformation using pollen or viruses,and chemicals that increase free DNA uptake, or free DNA delivery usingmicroprojectile bombardment. See, e.g., Narusaka, Y., et al., Chapter 9,in Transgenic Plants—Advances and Limitations, edited by Yelda, O., ISBN978-953-51-0181-9 (2012).

In some embodiments, a host cell is transiently or non-transientlytransfected. In some embodiments, a cell is transfected as it naturallyoccurs in a subject. In some embodiments, a cell that is transfected istaken from a subject, e.g., a primary cell or progenitor cell. In someembodiments, the primary cell or progenitor cell is cultured and/or isreturned after ex vivo transfection to the same subject (autologoustreatment) or to a different subject.

The NASC polynucleotide composition/first nucleic acid bindingprotein/second nucleic acid binding protein (comprising, for example,Class 2 CRISPR-Cas proteins) complexes described herein can be used togenerate non-human transgenic organisms by site-specifically introducinga selected polynucleotide sequence at a DNA target locus in the genometo generate a modification of the gDNA. The transgenic organism can bean animal or a plant.

A transgenic animal is typically generated by introducing the systeminto a zygote cell. A basic technique, described with reference tomaking transgenic mice (Cho, A., et al., “Generation of TransgenicMice,” Current Protocols in Cell Biology, CHAPTER.Unit-19.11 (2009)),involves five basic steps: first, preparation of a system, as describedherein, including a suitable donor polynucleotide; second, harvesting ofdonor zygotes; third, microinjection of the system into the mousezygote; fourth, implantation of microinjected zygotes intopseudo-pregnant recipient mice; and fifth, performing genotyping andanalysis of the modification of the gDNA established in founder mice.The founder mice will pass the genetic modification to any progeny. Thefounder mice are typically heterozygous for the transgene. Matingbetween these mice will produce mice that are homozygous for thetransgene 25% of the time.

Methods for generating transgenic plants are also well known. Atransgenic plant generated, e.g., using Agrobacterium transformationmethods, typically contains one transgene inserted into one chromosome.It is possible to produce a transgenic plant that is homozygous withrespect to a transgene by sexually mating (i.e., selfing) an independentsegregant transgenic plant containing a single transgene to itself, forexample an F0 plant, to produce F1 seed. Plants formed by germinating F1seeds can be tested for homozygosity. Typical zygosity assays include,but are not limited to, single nucleotide polymorphism assays andthermal amplification assays that distinguish between homozygotes andheterozygotes.

As an alternative to using a system described herein for the directtransformation of a plant, transgenic plants can be formed by crossing afirst plant that has been transformed with a system with a second plantthat has never been exposed to the system. For example, a first plantline containing a transgene can be crossed with a second plant line tointrogress the transgene into the second plant line, thus forming asecond transgenic plant line.

Further aspects of the present invention relate to methods of usingnucleoprotein compositions comprising NASC polynucleotide compositionsand a nucleic acid binding protein (e.g., Class 2 CRISPR-Cas proteins)complexes. Embodiments of such nucleoprotein compositions are describedherein. Numerous uses of the engineered nucleic acid sequences describedherein, include, but are not limited to, forming a scaffold of a complexof two or more engineered nucleic acid sequences comprising a nucleicacid binding Class 2 CRISPR protein binding sequence and a spacernucleic acid sequence complementary to a target nucleic acid sequence;precise editing of gDNA regions (e.g., excision, insertion,modification); tethering a donor polynucleotide in close proximity to acut-site (e.g., cleavage using a Cas9 protein or a Cpf1 protein);excision of a gDNA region and simultaneous donor polynucleotidetethering at the excision site; forming an artificial histone orintroduction of heterochromatin structure, for example, using dCas9; andtight transcriptional control of gene expression (e.g., blockingtranscription of a gene). Additional uses of the engineered nucleic acidsequence scaffolds described herein include, but are not limited to,methods of use and methods of manufacturing nucleoprotein particlesheets; flexible biomaterials, for example, for use in tissueengineering; caged drug delivery vehicles; vaccine delivery vehicles,for example, DNA or RNA vaccines; size-gated porous membranes, forexample, making and using membranes having holes of fixed size;nanoparticles of selected sizes; and protein nucleic acid polymers.

In one embodiment, the present invention includes a method of binding anucleic acid sequence (e.g., DNA) comprising contacting a first nucleicacid target sequence in the nucleic acid (e.g., DNA) and a secondnucleic acid target sequence in the nucleic acid sequence (e.g., DNA)with a NASC polynucleotide composition/first nucleic acid bindingprotein/second nucleic acid binding protein composition (comprising, forexample, Class 2 CRISPR-Cas proteins), thereby facilitating binding ofthe nucleoprotein to the first nucleic acid target sequence in thenucleic acid sequence and the second nucleic acid target sequence in thenucleic acid. The NASC polynucleotide composition/first nucleic acidbinding protein/second nucleic acid binding protein composition(comprising, for example, Class 2 CRISPR-Cas proteins) comprises a firstspacer element that is complementary to the first nucleic acid targetsequence (e.g., DNA) and a second spacer element that is complementaryto the second nucleic acid target sequence (e.g., DNA). In someembodiments, the nucleic acid target sequence is gDNA. Such methods ofbinding a nucleic acid target sequence can be carried in vitro (abiochemical assay), in cell (in cultured cells), ex vivo (cells removedfrom a subject), or in vivo (cells in an organism).

A variety of methods are known in the art to evaluate and/or quantitateprotein-nucleic acid interactions including, but not limited to, thefollowing: immunoprecipitation (ChIP) assays, DNA electrophoreticmobility shift assays (EMSA), DNA pull-down assays, and microplatecapture and detection assays. Commercial kits, materials, and reagentsare available to practice many of these methods from, for example,Thermo Scientific (Wilmington, Del.), Signosis (Santa Clara, Calif.),Bio-Rad (Hercules, Calif.), and Promega (Madison, Wis.). A commonapproach to detect protein-nucleic acid interactions is EMSA (see, e.g.,Hellman L. M., et al., Nature Protocols 2(8):1849-1861 (2007)).

In another embodiment, the present invention includes a method ofcutting a nucleic acid sequence (e.g., DNA) comprising contacting afirst nucleic acid target sequence in the nucleic acid (e.g., DNA) and asecond nucleic acid target sequence in the nucleic acid sequence (e.g.,DNA) with a nucleoprotein composition comprising a NASC polynucleotidecomposition/first nucleic acid binding protein/second nucleic acidbinding protein composition (comprising, for example, Class 2 CRISPR-Casproteins), thereby facilitating binding of the nucleoprotein compositionto the first nucleic acid target sequence in the nucleic acid sequenceand the second nucleic acid target sequence in the nucleic acid. Thenucleoprotein composition comprises a first spacer element that iscomplementary to the first nucleic acid target sequence (e.g., DNA) anda second spacer element that is complementary to the second nucleic acidtarget sequence (e.g., DNA). The first nucleic acid binding protein(e.g., Class 2 CRISPR-Cas protein) of the bound nucleoproteincomposition cuts the first nucleic acid target sequence, and the secondnucleic acid binding protein (e.g., Class 2 CRISPR-Cas protein) of thebound nucleic acid/protein composition cuts the second nucleic acidtarget sequence. In some embodiments, the nucleic acid target sequenceis gDNA. Such methods of binding a nucleic acid target sequence can becarried in vitro, in cell, ex vivo, or in vivo.

Methods of binding and of binding and cutting nucleic acid targetsequences using a NASC-PC1/NASC-PC2/S. thermophilus Cas9 protein/S.pyogenes protein composition are exemplified in FIG. 16A, FIG. 16B, andFIG. 16C. FIG. 16A illustrates a S. pyogenes Cas9 protein (FIG. 16A,1604) and a S. thermophilus Cas9 protein (FIG. 16A, 1603), aNASC-PC1/NASC-PC2 composition (FIG. 16A, 1600) (generally having thestructure shown in FIG. 6K), a double-stranded nucleic acid (FIG. 16A,1605) comprising a first DNA target binding sequence complementary tothe NASC-PC1/NASC-PC2 S. pyogenes Cas9 spacer element (FIG. 16A, 1602),and a double-stranded nucleic acid (FIG. 16A, 1607) comprising a secondDNA target binding sequence complementary to the NASC-PC1/NASC-PC2 S.thermophilus Cas9 spacer element (FIG. 16A, 1601). FIG. 16A, 1606,indicates the location of the S. pyogenes Cas9 PAM. FIG. 16A, 1608,indicates the location of the S. thermophilus Cas9 PAM.

FIG. 16A illustrates the formation of the S. pyogenes Cas9 protein (FIG.16A, 1604; complex 1609) and S. thermophilus Cas9 protein (FIG. 16A,1603; complex 1616) in complex with the NASC-PC1/NASC-PC2 composition(FIG. 11A, 1600; complex 1610).

FIG. 16A illustrates hydrogen bonding of the nucleoprotein complex tothe double-stranded DNA target sequences (FIG. 16A, 1611). FIG. 16A,1611, illustrates the binding of the NASC-PC1/NASC-PC2/S. thermophilusCas9 protein/S. pyogenes Cas9 protein composition to the double-strandednucleic acid (FIG. 16A, 1605) comprising a first DNA target bindingsequence complementary to the NASC-PC1/NASC-PC2 S. pyogenes Cas9 spacerelement (FIG. 16A, 1602) and a double-stranded nucleic acid (FIG. 16A,1607) comprising a second DNA target binding sequence complementary tothe NASC-PC1/NASC-PC2 S. thermophilus Cas9 spacer element (FIG. 16A,1601). If the S. pyogenes Cas9 protein and the S. thermophilus Cas9protein are enzymatically inactive, the nucleoprotein complex (FIG. 16A,1610) may be used, for example, to bring two DNA sequences (FIG. 16A,1605, 1607) into proximity (e.g., FIG. 16A, 1607).

FIG. 16B illustrates cleavage of the FIG. 16B, 1605, DNA by anenzymatically active S. pyogenes Cas9 protein and the tethering of theFIG. 16B, 1607, DNA using an enzymatically inactive S. thermophilus Cas9protein to maintain DNA (FIG. 16B, 1607) in proximity to the cleavagesite (FIG. 16B, 1612, 1613). Such a nucleoprotein complex may helpimprove the frequency of HDR using a donor polynucleotide (FIG. 16B,1607).

FIG. 16C illustrates cleavage of the FIG. 16C, 1605, DNA by anenzymatically active S. pyogenes Cas9 protein to break both strands ofthe FIG. 16C, 1605, DNA (FIG. 16C, 1612, 1613) and cleavage of the FIG.16C, 1607, DNA by an enzymatically active S. thermophilus Cas9 proteinto break both strands in the FIG. 16C, 1607, DNA (FIG. 16C, 1614, 1615).Such a nucleoprotein complex may be used to facilitate chromosomalrearrangement (e.g., translocations).

In yet another embodiment, the present invention includes a method ofmodifying DNA in a cell comprising contacting a first DNA targetsequence in the DNA and a second DNA target sequence in the DNA with aNASC polynucleotide composition/first nucleic acid bindingprotein/second nucleic acid binding protein composition (comprising, forexample, Class 2 CRISPR-Cas proteins such as Cas9 protein and/or Cpf1protein), thereby facilitating binding of the nucleoprotein complex tothe first nucleic acid target sequence in the nucleic acid sequence andthe second nucleic acid target sequence in the nucleic acid. The NASCpolynucleotide composition/first nucleic acid binding protein/secondnucleic acid binding protein composition (comprising, for example, Class2 CRISPR-Cas proteins) comprises a first spacer element that iscomplementary to the first nucleic acid target sequence and a secondspacer element that is complementary to the second nucleic acid targetsequence (e.g., DNA). The first protein of the bound nucleoproteincomplex cuts the first DNA target sequence, and the second protein ofthe bound nucleoprotein complex cuts the second DNA target sequence. Thecell repairs both the first cut site and the second cut site. Exemplarycell DNA repair pathways include HDR, NHEJ, and MMEJ. In someembodiments, the nucleic acid target sequence is gDNA. Such methods ofbinding a nucleic acid target sequence can be carried out in vitro, incell, ex vivo, or in vivo. The contracting step may further comprise adonor polynucleotide being present, wherein at least a portion of thedonor polynucleotide is incorporated between the first cut site and thesecond cut site.

In another embodiment, the invention relates to a method to bring adonor polynucleotide into proximity of a DSB in a nucleic acid target,typically DNA, in a cell. The method comprises contacting a first DNAtarget sequence in the DNA and a second DNA target sequence in a donorpolynucleotide with NASC polynucleotide composition/first DNA bindingprotein/second DNA binding protein composition (comprising, for example,Class 2 CRISPR-Cas proteins such as Cas9 protein and/or Cpf1 protein)having a first DNA target binding sequence complementary to the firstDNA target and a second DNA target binding sequence complementary to thesecond DNA target. The first DNA binding protein is catalytically activeand is associated with the first DNA target binding sequence. The secondDNA binding protein is enzymatically inactive and is associated with thesecond DNA target binding sequence. Contacting the nucleoprotein complexwith the first and second DNA target sequences facilitates the bindingof the nucleoprotein complex to the first DNA target sequence in the DNAand the second DNA target sequence in the donor polynucleotide. Thecatalytically active DNA binding protein of the nucleoprotein complexcuts the first DNA target sequence to form a cut site. The donorpolynucleotide is in proximity to the cut site (e.g., the DSB) becausethe catalytically active DNA binding protein and the catalyticallyinactive DNA binding protein are complexed with the NASC polynucleotidecomposition, that is, they are part of the same nucleoprotein complex.In some embodiments, at least a portion of the donor polynucleotide isintroduced into the cut site in the DNA (e.g., by an HDR repair process)resulting in modifying the DNA.

FIG. 12 illustrates using NASC-PC1/NASC-PC2/active Cas9 protein/dCas9Cas9 protein composition, wherein endonuclease domains of the activeCas9 are active and the endonuclease domains of the dCas9 are inactive,to bring a donor polynucleotide into proximity of a DSB in a nucleicacid target sequence. FIG. 12 illustrates the active Cas9 protein (FIG.12, 1211) and the dCas9 protein (FIG. 12, 1203), NASC-PC1/NASC-PC2composition (FIG. 12, 1205; see also FIG. 6F); a double-stranded nucleicacid (FIG. 12, 1206/1207) comprising a first DNA target binding sequencecomplementary to the active Cas9-NASC-PC1/NASC-PC2 composition spacerelement (FIG. 12, 1210); and a donor polynucleotide (FIG. 12, 1200/1201)comprising a second DNA target binding sequence complementary to thedCas9-NASC-PC1/NASC-PC2 composition spacer element (FIG. 12, 1204). FIG.12 illustrates the NASC-PC1/NASC-PC2/active Cas9 protein/dCas9 proteinin complex and the hydrogen bonding of the first DNA target bindingsequence to the first DNA target sequence upstream of a first Cas9 PAM(FIG. 12, 1209) and the second DNA target binding sequence to the secondtarget sequence upstream of a second Cas9 PAM (FIG. 12, 1202) in thedonor polynucleotide. FIG. 12, 1208, illustrates double-strand blunt-endcuts made by Cas9 at the first DNA target binding sequence, resulting ina second double-stranded nucleic acid (FIG. 12, 1213/1212), and showsthe donor polynucleotide (FIG. 12, 1200/1201) in proximity to thedouble-strand blunt-end cuts. Having the donor polynucleotide in closeproximity to the double-strand cuts increases the likelihood ofintegration of the donor polynucleotide sequences, or portions thereof,into the DNA comprising the first nucleic acid target.

In a further embodiment, the invention relates to a method bringing afirst nucleic acid target site, typically DNA, into the proximity of asecond nucleic acid target site, typically DNA, in a cell. The methodcomprises contacting a first nucleic target sequence and a secondnucleic target sequence with a nucleoprotein complex comprising NASCpolynucleotide composition in a complex with a nucleic acid bindingprotein, and a second nucleic acid binding protein, thereby facilitatingbinding of the nucleoprotein complex to the first nucleic acid targetsequence and the second nucleic acid target sequence. The first DNAtarget sequence is complementary to a first nucleic acid bindingsequence of the NASC polynucleotide composition, wherein the associatedfirst protein is a catalytically inactive nucleic acid binding protein(e.g., a dCpf1 protein or a dCas9 protein). The second DNA targetsequence is complementary to a second nucleic acid binding sequence ofthe NASC polynucleotide composition, wherein the associated secondprotein is a catalytically inactive nucleic acid binding protein (e.g.,a dCpf1 protein or a dCas9 protein). The first nucleic acid target siteis brought into proximity of a second nucleic acid target site becausethe first and second catalytically inactive nucleic acid bindingproteins are complexed with the NASC polynucleotide composition, thatis, they are part of the same nucleic acid/protein composition. In someembodiments, the first nucleic acid target sequence and the secondnucleic acid target sequence are on separate polynucleotides (e.g.,different chromosomes) or a single polynucleotide comprises the firstnucleic acid target sequence and the second nucleic acid target sequence(e.g., different sections of the same chromosome).

FIG. 13 illustrates an example of a NASC polynucleotide composition/afirst dCas9 protein/second dCas9 binding protein/a third dCas9 proteincomposition binding to three sites within a single DNA polynucleotide.The NASC polynucleotide composition is also illustrated in FIG. 6I. Thisnucleoprotein complex can be used to in a method of bringing a firstnucleic acid target site, typically DNA, into the proximity of a secondnucleic acid target site, typically DNA, into the proximity of a thirdnucleic acid target site, typically DNA, in a cell. This method also canbe applied, for example, to detection of nucleic acid target sites inproximity and modulating in vitro or in vivo transcriptional modulationof a gene adjacent the three target sites. Indicators of the componentsillustrated in FIG. 13 are presented in Table 8.

FIG. 14 illustrates an example of a NASC polynucleotide composition/afirst dCas9 protein/second active Cas9 binding protein/a third activeCas9 protein composition binding to three sites of multiple DNApolynucleotides. The NASC polynucleotide composition is also illustratedin FIG. 6I. This nucleoprotein complex can be used, for example, in amethod to bring a donor polynucleotide into proximity of two DSBs in anucleic acid target, typically DNA, in a cell to facilitate HDRintegration of the donor polynucleotide or portions of the donorpolynucleotide into the region between the two DNA target cleavagesites. Indicators of the components illustrated in FIG. 14 are presentedin Table 8. The NASC polynucleotide composition is also illustrated inFIG. 6I.

FIG. 15 illustrates an example of a NASC polynucleotide composition/afirst dCas9 protein/second active Cas9 binding protein/a third activeCas9 protein composition binding to three sites in three different DNApolynucleotides. The NASC polynucleotide composition is also illustratedin FIG. 6I. This nucleoprotein complex can be used, for example, in amethod to improve ligation frequency of two DNA polynucleotides to the5′ and 3′ ends of a third DNA polynucleotide. Indicators of thecomponents illustrated in FIG. 15 are presented in Table 8. The NASCpolynucleotide composition is also illustrated in FIG. 6I.

TABLE 8 Indicators and Corresponding Regions for FIG. 13, FIG. 14, andFIG. 15 FIG. 13 FIG. 14 FIG. 15 Indi- Indi- Indi- cator Component catorComponent cator Component 1300 a 3′ end of 1400 a 3′ end of 1500 a 3′end of a first strand a first strand a first strand of a DNA of a firstDNA of a first DNA 1301 a 5′ end of 1401 a 3′ end of 1501 a 3′ end of afirst strand a second strand a second strand of a DNA of a first DNA ofa first DNA 1302 a first Cas9 1402 a first Cas9 1502 a first Cas9protein protein protein 1303 a first DNA 1403 a first DNA 1503 a firstDNA target binding target binding target binding sequence sequencesequence 1304 a first 1404 a first 1504 a first nucleic acid nucleicacid nucleic acid binding protein binding protein binding proteinbinding binding binding sequence sequence sequence 1305 a first 1405 afirst 1505 a first Cas9 PAM Cas9 PAM Cas9 PAM 1306 a second 1408 asecond 1508 a second Cas9 protein Cas9 protein Cas9 protein 1307 asecond 1411 a second 1511 a second Cas9 PAM Cas9 PAM Cas9 PAM 1308 asecond 1409 a second 1509 a second DNA target DNA target DNA targetbinding binding binding sequence sequence sequence 1309 a second 1410 asecond DNA 1510 a second DNA nucleic acid target binding target bindingbinding protein sequence sequence binding protein binding proteinbinding sequence sequence sequence 1310 a 3′ end of 1406/ 5′/3′ ends of1507/ 5′/3′ ends of a first strand 1412 a first strand 1506 a second ofa DNA of a second DNA DNA 1311 a 5′ end of 1413/ 5′/3′ends of 1514/5′/3′ ends of a first strand 1407 a second strand 1513 a third of a DNAof a second DNA DNA 1312 a third 1414 a third 1515 a third Cas9 proteinCas9 protein Cas9 protein 1313 a third 1417 a third 1518 a third Cas9PAM Cas9 PAM Cas9 PAM 1314 a third 1416 a third 1517 a third nucleicacid nucleic acid nucleic acid binding protein binding protein bindingprotein binding binding binding sequence sequence sequence 1315 a third1415 a third 1516 a third nucleic acid nucleic acid nucleic acid targetbinding target binding target binding sequence sequence sequence a first1418 a double- 1512 a double- double- strand break strand break strandbreak in the second in the second in the second DNA DNA DNA a second1419 a double- 1519 a double- double- strand break strand break strandbreak in the third in the third in the second DNA DNA DNA

In yet another embodiment, the present invention also includes methodsof modulating in vitro or in vivo transcription, for example,transcription of a gene comprising regulatory element sequences. Themethod comprises contacting at least a first nucleic target sequence anda second nucleic target sequence with a NASC polynucleotidecomposition/first nucleic acid binding protein/second nucleic acidbinding protein composition (comprising, for example, catalyticallyinactive Class 2 CRISPR-Cas proteins such as dCas9 and/or dCpf1),thereby facilitating binding of the nucleoprotein composition to thefirst nucleic acid target sequence and the second nucleic acid targetsequence. At least one of the first DNA target sequence and the secondDNA target sequence comprises the regulatory element sequences. Thefirst DNA target binding sequence of the NASC polynucleotidecomposition/first nucleic acid binding protein/second nucleic acidbinding protein composition is complementary to a first nucleic acidtarget sequence. The second DNA target binding sequence of the NASCpolynucleotide composition/first nucleic acid binding protein/secondnucleic acid binding protein composition is complementary to a secondDNA target sequence. In addition, the first and/or second protein can befusion proteins, for example, dCas9 fused to a repressor or activatordomain, and/or dCpf1 fused to a repressor or activator domain. Thebinding of the nucleic acid/protein composition to the first DNA targetsequence and the second DNA target sequence modulates transcription ofthe gene. In some embodiments, the first DNA target sequence and thesecond DNA target sequence comprise the regulatory element sequences,and the first DNA target sequence comprises a promoter and the secondDNA target sequence comprises a transcription start site.

FIG. 11 illustrates a method of modulating in vitro or in vivotranscription using NASC polynucleotide compositions of the presentinvention. In this figure, a NASC polynucleotide composition/a firstdCas9 protein/a second dCsa9 protein complex is formed (FIG. 11, 1111,1110, 1103) by the association of a NASC polynucleotide composition(FIG. 11, 1103) with a first dCas9 protein (FIG. 11, 1100) and a seconddCsa9 protein (FIG. 11, 1101). The complex comprises a first DNA targetbinding sequence (FIG. 11, 1102) complementary to a first nucleic targetsequence that is adjacent a first Cas9 PAM (FIG. 11, 1108) and a secondDNA target binding sequence (FIG. 11, 1104) that is complementary to asecond nucleic target sequence that is adjacent a second Cas9 PAM (FIG.11, 1109) in a DNA polynucleotide (FIG. 11, 1105). The NASCpolynucleotide composition/first dCas9 protein/second dCsa9 proteincomplex is contacted with the DNA polynucleotide comprising the DNAtarget sequences, thereby facilitating binding of the nucleoproteincomposition through hydrogen-bonded base pairs (FIG. 11, 1112, 1113) tothe first nucleic acid target sequence and the second nucleic acidtarget sequence. At least one of the first DNA target sequence and thesecond DNA target sequence comprise the regulatory element sequences.The first DNA target binding sequence of the NASC polynucleotidecomposition/first dCas9 protein/second dCsa9 protein composition iscomplementary to a first nucleic acid target sequence. The second DNAtarget binding sequence of the NASC polynucleotide composition/a firstdCas9 protein/a second dCsa9 protein composition is complementary to asecond DNA target sequence.

The NASC polynucleotide compositions of the present invention can beused to design nucleic acid/protein macromolecules that self-assembleinto complex architectures. Such macromolecules have many uses innanobiotechnology including, but not limited to, drug delivery, designof nucleic acid/protein nanomaterials, and formation of nanostructuressuch as nanotubes and closed-cage structures. Example 13 illustrates theuse of NASC polynucleotide compositions of the present invention forformation of NASC closed-cage compositions (NASC-CCs). NASC-CCs may beused for packaging of small molecules. Example 14 describes methods thatcan be used for characterization of NASC-CC/dCas protein complexes toverify proper assembly and assess the size and volume of assembledNASC-CC/dCas protein complexes. The NASC-CC described in Example 13 andExample 14 is illustrated in FIG. 6L. Two NASC polynucleotidecompositions corresponding to the NASC polynucleotide compositionillustrated in FIG. 6A (referred to in the Example as aNASC-PC1-triplex) can be connected using double-stranded DNA bracenucleic acid sequences. The double-stranded DNA brace nucleic acidsequence can comprise a first DNA target sequence and a second DNAtarget sequence. As described in Example 13, the NASC-CC isself-assembling because a first NASC-PC1-triplex/dCas9 proteinnucleoprotein complex comprises DNA target binding sequences that willspecifically bind the first DNA target sequence of the brace nucleicacid sequence. A second NASC-PC1-triplex/dCas9 protein comprising DNAtarget binding sequences will specifically bind the second DNA targetsequence of the brace nucleic acid sequence to form a closed cagestructure. FIG. 6M illustrates the NASC-CC with six associated Cas9proteins forming a nucleoprotein cage.

A wide variety of molecules are candidates for incorporation intoNASC-CC polynucleotide compositions to facilitate delivery of themolecules include, but not limited to, vaccines (e.g., inactivatedvaccines, attenuated vaccines, protein subunit vaccines, and nucleicacid vaccines); monoclonal antibodies; antibiotics; small moleculedrugs; cancer therapeutics; recombinant proteins, biologics, and thelike. Such molecules are also referred to herein as “payload.”

Fusions of targeting proteins and nucleic acid binding proteins (e.g.,Cas9, Cpf1) can be used to achieve tissue, organ, or cell type targeteddelivery of NASC-CC polynucleotide compositions. For example, landscapephage peptides specific for specific tumors can be obtained by affinityselection and purified peptides specific for specific tumors can befused to a Cas9 protein. The Cas9 fusion protein can then be used toassemble a NASC-CC polynucleotide composition to obtain tumor-targetednanocarriers. Production of phage peptides specific for specific tumorshas been described by Jayanna, P., et al., Nanomedicine. 5(1):83 (2009).

Alternative modes of delivery of NASC-CC to cells can be achievedthrough the linkage, packaging, or association of NASC-CC RNAs, DNAs, orproteins (“NASC-CC/Cas”) components with various ligands or chemicalagents. Packaging techniques include NASC-CC/Cas packaging intoself-assembling liposomes, micelles, dendrimers, nanospheres, ornanocapsules.

Covalent and noncovalent attachment of polyethelyne glycol (PEG;PEGylation) to molecules and macrostructures has been employed for thepackaging of payloads for target delivery to cells and can be adaptedfor the encapsulation of NASC-CC/Cas by one of ordinary skill in the artin view of the teachings of the present specification. Furthermore,protein PEGylation is a widely practiced form of conjugation chemistryfor delivery of macromolecules to tissues, cells, and organelles.PEGylated structures can be further modified with molecular attachmentof moieties that facilitate cellular uptake (e.g., a folate moiety).Selection of these moieties relies on the unique properties of cellstargeted for directed delivery of NASC-CC/Cas and the encapsulatedpayload (i.e., extracellular matrix, receptors, or antibodycomposition). These moieties can be attached to the NASC-CC/Cas, NASC-CCpackaging agent, or both the NASC-CC/Cas and NASC-CC packaging agent.Moieties that can be used include, but are not limited to, antibodies,ligands, transferrins, glycoproteins, aptamers, cell penetratingpeptides, matrix metalloprotease-cleavable peptides, integrins, proteintransduction domains, epitopes, cell adhesion molecules, and othercompounds known in the art (see, e.g., Steichen, S. et. al., EuropeanJournal of Pharmaceutical Sciences. 48(3):416-27 (2013); Dashpande, P.,et al., Nanomedicine. 8(9):1509-28 (2013)).

Trigger release of NASC-CC/Cas encapsulated agents can be facilitated bythe incorporation of distinct chemical moieties or sequence motifs intothe NASC-CC/Cas composition or within the NASC-CC packaging agent.Attachment of biodegradable polymeric compositions (e.g., a modified PEGcomposition) to a NASC-CC/Cas or a NASC-CC packaging agent can allow forthe breakdown of the NASC-CC/Cas or NASC-CC packaging agent uponcellular uptake. Engineered sensitive sites (i.e., proteolytic sensitivepeptide sequences, pH sensitive copolymers, redox sensitive linkages,etc.) or combinations of engineered sensitive sites may be employed tofacilitate release of NASC-CC encapsulated agents. Labile linkagesbetween the NASC-CC and NASC-CC packaging agent, such as a pH sensitivelinkages, can be utilized to encourage disassociation from the NASC-CCand the NASC-CC packaging agent in high pH environments (e.g., anendocytic vacuole). NASC-CC/Cas complexes can be further modified withorganelle specific epitopes (i.e., a nuclear localization signal) fordelivery of payload to specific organelles.

One of ordinary skill in the art, in view of the teachings of thespecification, can use a variety of different NASC polynucleotidecompositions to form a variety of nanostructures.

Any of the components of the nucleoprotein compositions comprising aNASC polynucleotide composition of the present invention or nucleic acidsequences encoding such components, as described above, can beincorporated into a kit, optionally including one or more reagents. Insome embodiments, a kit includes a package with one or more containersholding the kit elements, as one or more separate compositions or,optionally, as admixture wherein the compatibility of the componentswill allow. In some embodiments, kits also comprise a buffer, abuffering agent, a salt, a sterile aqueous solution, and/orpreservatives. Illustrative kits comprise one or more components of aNASC polynucleotide composition and optionally one or more cognatenucleic acid binding proteins, such as a Cpf1 and/or a Cas9 protein; andone or more nucleic acid sequences encoding one or more components of aNASC polynucleotide composition, and optionally one or more nucleic acidsequences encoding a Cpf1 and/or a Cas9 protein.

Furthermore, kits can further comprise instructions for using componentsof the nucleoprotein complexes comprising NASC polynucleotidecompositions of the present invention or nucleic acid sequences encodingsuch components. Instructions included in kits of the invention can beaffixed to packaging material or can be included as a package insert.Although the instructions are typically written or printed materials,they are not limited to such. Any medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), RF tags, and the like. Instructions can alsoinclude the address of an internet site that provides the instructions.

Another aspect of the invention relates to methods of making ormanufacturing a NASC polynucleotide composition or a nucleicacid/protein composition comprising a NASC polynucleotide composition ofthe present invention. In one embodiment, the methods of making ormanufacturing comprise chemically synthesizing polynucleotide componentsof a NASC polynucleotide composition. In some embodiments, a NASCpolynucleotide composition comprises RNA bases and can be generated fromDNA templates using in vitro transcription.

In some embodiments, NASC polynucleotide composition components can bemodified by a moiety (e.g., a ligand moiety, a ligand binding moiety, anaffinity tag, an exonuclease resistance moiety). Polynucleotidecomponents can be connected to, for example, the 5′ terminal sequenceand/or 3′ terminal sequence of a polynucleotide component.

A nucleic acid/protein composition comprising NASC polynucleotidecomposition can further comprise a detectable label, including a moietythat can provide a detectable signal. Examples of detectable labelsinclude, but are not limited to, an enzyme, a radioisotope, a member ofa specific binding pair, a fluorophore (FAM), a fluorescent protein(green fluorescent protein, red fluorescent protein, mCherry, tdTomato),an DNA or RNA aptamer together with a suitable fluorophore (enhanced GFP(EGFP), “Spinach”), a quantum dot, an antibody, and the like. A largenumber and variety of suitable detectable labels are well-known to oneof ordinary skill in the art.

A nucleic acid/protein composition comprising a NASC polynucleotidecomposition or cells modified by use of a nucleic acid/proteincomposition comprising NASC polynucleotide composition, as describedherein, can be used as a pharmaceutical composition formulated, forexample, with a pharmaceutically acceptable excipient. Illustrativeexcipients include carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and the like. The pharmaceuticalcomposition can facilitate administration of a nucleic acid/proteincomposition comprising an engineered NASC polynucleotide composition toan organism. Pharmaceutical compositions can be administered intherapeutically effective amounts by various forms and routes including,for example, intravenous, subcutaneous, intramuscular, oral, aerosol,parenteral, ophthalmic, and pulmonary administration.

Numerous advantages may be obtained using the NASC polynucleotidecompositions and nucleoprotein complexes of the present inventionincluding, but not limited to, the following:

-   -   reduction in off-targeting binding using a nucleic acid/protein        composition comprising nucleic acid binding proteins (e.g.,        Class 2 CRISPR-Cas proteins) and a NASC polynucleotide        composition that targets binding to multiple target nucleic acid        sequences using a single nucleoprotein complex relative to use        of similarly targeted individual NATNA/nucleic acid binding        protein complexes (e.g., a sgRNA/Cas9 protein complex);    -   tethering of a donor polynucleotide through use of a nucleic        acid/protein composition comprising nucleic acid binding        proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition to bring the donor polynucleotide        into proximity of a cut in a double-stranded nucleic acid;    -   bringing two separate polynucleotides (e.g., two different        chromosomes) or two regions of a single polynucleotide (e.g.,        two regions of a single chromosome) into proximity of each other        using a nucleic acid/protein composition comprising nucleic acid        binding proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition;    -   transcriptional modulation of a target gene by binding of a        nucleic acid/protein composition comprising nucleic acid binding        proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition to multiple regulatory sequences        operably linked to the target gene;    -   transcriptional modulation of a target gene by binding of a        nucleic acid/protein composition comprising nucleic acid binding        proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition to bring two separate polynucleotides        (e.g., trans-acting regulatory element) or two regions of a        single polynucleotide (e.g., cis-acting regulatory element) into        proximity of each other;    -   simultaneous targeting of multiple target nucleic acid sequences        using a nucleic acid/protein composition comprising nucleic acid        binding proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition, including embodiments wherein a        donor polynucleotide is also tethered to the nucleic        acid/protein composition;    -   forming biological nanostructures comprising a nucleic        acid/protein composition comprising nucleic acid binding        proteins (e.g., Class 2 CRISPR-Cas proteins) and a NASC        polynucleotide composition, for example, for pharmaceutical        formulation of small molecules;    -   building nanoscale architectures with nucleic acid/protein        compositions comprising nucleic acid binding proteins (e.g.,        Class 2 CRISPR-Cas proteins) and NASC polynucleotide        compositions that have predefined sizes and shapes; and    -   designing nucleic acid/protein components, comprising nucleic        acid/protein compositions comprising nucleic acid binding        proteins (e.g., Class 2 CRISPR-Cas proteins) and NASC        polynucleotide compositions, that self-assemble into        predetermined complex architectures.

Various embodiments contemplated herein include, but are not limited to,one or more of the following. The embodiments are numbered for ease ofreference.

Embodiments of the present invention include, but are not limited to,the following.

1. A complex of two or more engineered nucleic acid sequences forming ascaffold, comprising: a first engineered nucleic acid comprising—a firstelement 1 comprising a first double-stranded nucleic acid bindingprotein binding sequence having a first end and a second end—a secondelement 1 comprising a repeat nucleic acid sequence 1, wherein therepeat nucleic acid sequence 1 is proximal to the first end of the firstdouble-stranded nucleic acid binding protein binding sequence—a thirdelement 1 comprising a nucleic acid sequence 1;—and a second engineerednucleic acid comprising, a first element 2 comprising a seconddouble-stranded nucleic acid binding protein binding sequence, having afirst end and a second end—a second element 2 comprising a repeatnucleic acid sequence 1C, wherein the repeat nucleic acid sequence 1C isproximal to the first end of the first double-stranded nucleic acidbinding protein binding sequence—and a third element 2 comprising anucleic acid sequence 2; wherein the repeat nucleic acid sequence 1 isassociated with the repeat nucleic acid sequence 1C through hydrogenbonding between the repeat nucleic acid sequence 1 and the repeatnucleic acid sequence 1C.

2. The complex of embodiment 1, wherein the first engineered nucleicacid comprises—the first element 1 further comprising—the firstdouble-stranded nucleic acid binding protein binding sequence, whereinthe first end is a 5′ end and the second end is a 3′ end—the secondelement 1 further comprising the repeat nucleic acid sequence 1 having a5′ end and a 3′ end, wherein the 3′ end of the repeat nucleic acidsequence 1 is located 5′ of the 5′ end of the first double-strandednucleic acid binding protein binding sequence—and the third element 1further comprising the nucleic acid sequence 1, having a 5′ end and a 3′end, wherein the 5′ end of the nucleic acid sequence 1 is located 3′ ofthe 3′ end of the first double-stranded nucleic acid binding proteinbinding sequence; and—the second engineered nucleic acid comprises, thefirst element 2 further comprising the second double-stranded nucleicacid binding protein binding sequence, wherein the first end is a 5′ endand the second end is a 3′ end—the second element 2 further comprisingthe repeat nucleic acid sequence 1C having a 5′ end and a 3′ end,wherein the 3′ end of the repeat nucleic acid sequence 1C is located 5′of the 5′ end of the 5′ end of the second double-stranded nucleic acidbinding protein binding sequence—and the third element 2 furthercomprising the nucleic acid sequence 2 has a 5′ end and a 3′ end,wherein the 5′ end of the nucleic acid sequence 2 is located 3′ of the3′ end of the second double-stranded nucleic acid binding proteinbinding sequence.

3. The complex of embodiment 1, wherein the first engineered nucleicacid comprises,—the first element 1 further comprising the firstdouble-stranded nucleic acid binding protein binding sequence, whereinthe first end is a 5′ end and the second end is a 3′ end—the secondelement 1 further comprising the repeat nucleic acid sequence 1, havinga 5′ end and a 3′ end, wherein the 3′ end of the repeat nucleic acidsequence 1 is located 5′ of the 5′ end of the first double-strandednucleic acid binding protein binding sequence—and the third element 1further comprising the nucleic acid sequence 1, having a 5′ end and a 3′end, wherein the 3′ end of the nucleic acid sequence 1 is located 5′ ofthe 5′ end of the repeat nucleic acid sequence 1; and the secondengineered nucleic acid comprises—the first element 2 further comprisingthe second double-stranded nucleic acid binding protein bindingsequence, wherein the first end is a 5′ end and the second end is a 3′end—the second element 2 further comprising the repeat nucleic acidsequence 1C having a 5′ end and a 3′ end, wherein the 3′ end of therepeat nucleic acid sequence 1C is located 5′ of the 5′ end of the 5′end of the second double-stranded nucleic acid binding protein bindingsequence—and the third element 2 further comprising the nucleic acidsequence 2 has a 5′ end and a 3′ end, wherein the 3′ end of the nucleicacid sequence 2 is located 5′ of the 5′ end of the repeat nucleic acidsequence 1C.

4. A complex of two or more engineered nucleic acid sequences forming ascaffold, comprising: a first engineered nucleic acid sequencecomprising, a first element 1 comprising a first nucleic acid bindingClass 2 CRISPR protein binding sequence, having a first end and a secondend—a second element 1 comprising a repeat nucleic acid sequence 1,wherein the repeat nucleic acid sequence 1 is proximal to the first endof the first nucleic acid binding Class 2 CRISPR protein bindingsequence—and a third element 1 comprising a nucleic acid sequence 1;—anda second engineered nucleic acid sequence comprising, a first element 2comprising a second nucleic acid binding Class 2 CRISPR protein bindingsequence, having a first end and a second end—a second element 2comprising a repeat nucleic acid sequence 1C, wherein the repeat nucleicacid sequence 2 is proximal to the first end of the second nucleic acidbinding Class 2 CRISPR protein binding sequence—and a third element 2comprising a nucleic acid sequence 2; wherein the repeat nucleic acidsequence 1 is associated with the repeat nucleic acid sequence 1Cthrough hydrogen bonding between the repeat nucleic acid sequence 1 andthe repeat nucleic acid sequence 1C.

5. The complex of embodiment 4, wherein the first nucleic acid bindingClass 2 CRISPR protein binding sequence is a Class 2 Type V CRISPRprotein binding sequence, wherein the first end is a 5′ end and thesecond end is a 3′ end—the repeat nucleic acid sequence 1 has a 5′ endand a 3′ end, wherein the 3′ end of the repeat nucleic acid sequence 1is located 5′ of the 5′ end of the first nucleic acid binding Class 2Type V CRISPR protein binding sequence—and the nucleic acid sequence 1has a 5′ end and a 3′ end, wherein the 5′ end of the nucleic acidsequence 1 is located 3′ of the 3′ end of the first nucleic acid bindingClass 2 Type V CRISPR protein binding sequence;—and the second nucleicacid binding Class 2 CRISPR protein binding sequence is a Class 2 Type VCRISPR protein binding sequence, wherein the first end is a 5′ end andthe second end is a 3′ end—the repeat nucleic acid sequence 2 has a 5′end and a 3′ end, wherein the 3′ end of the repeat nucleic acid sequence2 is located 5′ of the 5′ end of the second nucleic acid binding Class 2Type V CRISPR protein binding sequence—and the nucleic acid sequence 2has a 5′ end and a 3′ end, wherein the 5′ end of the nucleic acidsequence 2 is located 3′ of the 3′ end of the second nucleic acidbinding Class 2 Type V CRISPR protein binding sequence.

6. The complex of embodiment 5, wherein the repeat nucleic acid sequence1 further comprises a linker element nucleic acid sequence 1-1, having a5′ end and a 3′ end—a repeat nucleic acid sequence 1a, having a 5′ endand a 3′ end—a linker element nucleic acid sequence 1-2, having a 5′ endand a 3′ end—a repeat nucleic acid sequence 1b, having a 5′ end and a 3′end—and a linker element nucleic acid sequence 1-3, having a 5′ end anda 3′ end, arranged in the following 3′ to 5′ order: the linker elementnucleic acid sequence 1-1, the repeat nucleic acid sequence 1a, thelinker element nucleic acid sequence 1-2, the repeat nucleic acidsequence 1b, and the linker element nucleic acid sequence 1-3;—and therepeat nucleic acid sequence 2 further comprises a linker elementnucleic acid sequence 2-1, having a 5′ end and a 3′ end—a repeat nucleicacid sequence 1bC, having a 5′ end and a 3′ end—a linker element nucleicacid sequence 2-2, having a 5′ end and a 3′ end—a repeat nucleic acidsequence 2a, having a 5′ end and a 3′ end—and a linker element nucleicacid sequence 2-3, having a 5′ end and a 3′ end, arranged in thefollowing 3′ to 5′ order: the linker element nucleic acid sequence 2-1,the repeat nucleic acid sequence 1bC, the linker element nucleic acidsequence 2-2, the repeat nucleic acid sequence 2a, and the linkerelement nucleic acid sequence 2-3; wherein the repeat nucleic acidsequence 1 is associated with the repeat nucleic acid sequence 2 throughhydrogen bonding between the repeat nucleic acid sequence 1b and therepeat nucleic acid sequence 1bC.

7. The complex of embodiment 6, further comprising a third engineerednucleic acid comprising, a first element 3 comprising a third nucleicacid binding Class 2 Type V CRISPR protein binding sequence, wherein thefirst end is a 5′ end and the second end is a 3′ end—and a secondelement 3 comprising a repeat nucleic acid sequence 3 having a 5′ endand a 3′ end, wherein the 3′ end of the repeat nucleic acid sequence 3is located 5′ of the 5′ end of the third nucleic acid binding Class 2Type V CRISPR protein binding sequence, wherein the repeat nucleic acidbinding sequence 3 further comprises a linker element nucleic acidsequence 3-1, having a 5′ end and a 3′ end—a repeat nucleic acidsequence 2aC, having a 5′ end and a 3′ end—a linker element nucleic acidsequence 3-2, having a 5′ end and a 3′ end—a repeat nucleic acidsequence 3a, having a 5′ end and a 3′ end—and a linker element nucleicacid sequence 3-3, having a 5′ end and a 3′ end, arranged in thefollowing 3′ to 5′ order: the linker element nucleic acid sequence 3-1,the repeat nucleic acid sequence 2aC, the linker element nucleic acidsequence 3-2, the repeat nucleic acid sequence 3a, and the linkerelement nucleic acid sequence 3-3;—and a third element 3 comprising anucleic acid sequence 3, having a 5′ end and a 3′ end, wherein the 5′end of the nucleic acid sequence 3 is located 3′ of the 3′ end of thefirst nucleic acid binding Class 2 Type V CRISPR protein bindingsequence;—and a fourth engineered nucleic acid comprising, a firstelement 4 comprising a fourth nucleic acid binding Class 2 Type V CRISPRprotein binding sequence, wherein the first end is a 5′ end and thesecond end is a 3′ end—a second element 4 comprising a repeat nucleicacid sequence 4 having a 5′ end and a 3′ end, wherein the 3′ end of therepeat nucleic acid sequence 3 is located 5′ of the 5′ end of the fourthnucleic acid binding Class 2 Type V CRISPR protein binding sequence,wherein the repeat nucleic acid binding sequence 4 further comprises alinker element nucleic acid sequence 4-1, having a 5′ end and a 3′ end—arepeat nucleic acid sequence 3aC, having a 5′ end and a 3′ end—a linkerelement nucleic acid sequence 4-2, having a 5′ end and a 3′ end—a repeatnucleic acid sequence 1aC, having a 5′ end and a 3′ end—and a linkerelement nucleic acid sequence 4-3, having a 5′ end and a 3′ end,arranged in the following 3′ to 5′ order: the linker element nucleicacid sequence 4-1, the repeat nucleic acid sequence 3aC, the linkerelement nucleic acid sequence 4-2, the repeat nucleic acid sequence 1aC,and the linker element nucleic acid sequence 4-3;—and the third element4 further comprising the nucleic acid sequence 4, having a 5′ end and a3′ end, wherein the 5′ end of the nucleic acid sequence 4 is located 3′of the 3′ end of the first nucleic acid binding Class 2 Type V CRISPRprotein binding sequence; wherein the repeat nucleic acid sequence 1 isassociated with the repeat nucleic acid sequence 2 through hydrogenbonding between the repeat nucleic acid sequence 1b and the repeatnucleic acid sequence 1bC, the repeat nucleic acid sequence 1 isassociated with the repeat nucleic acid sequence 4 through hydrogenbonding between the repeat nucleic acid sequence 1a and the repeatnucleic acid sequence 1aC, the repeat nucleic acid sequence 2 isassociated with the repeat nucleic acid sequence 3 through hydrogenbonding between the repeat nucleic acid sequence 2a and the repeatnucleic acid sequence 2aC, and the repeat nucleic acid sequence 3 isassociated with the repeat nucleic acid sequence 4 through hydrogenbonding between the repeat nucleic acid sequence 3a and the repeatnucleic acid sequence 3aC.

8. The complex of any one of embodiments 4 to 7, wherein the repeatnucleic acid sequence 1 and the repeat nucleic acid sequence 2 furthercomprise a double-stranded nucleic acid binding protein binding site 1and the double-stranded nucleic acid binding protein binding site 1 isformed by hydrogen base-pair bonding between the repeat nucleic acidsequence 1 and the repeat nucleic acid sequence 2.

9. The complex of embodiment 8, wherein the double-stranded nucleic acidbinding protein binding site 1 is a Csy4 protein binding site.

10. The complex of any one of embodiments 4 to 9, wherein the firstengineered nucleic acid and the second engineered nucleic acid eachcomprises RNA, DNA, or a combination thereof.

11. The complex of embodiment 7, wherein the first engineered nucleicacid, the second engineered nucleic acid, the third engineered nucleicacid, and the fourth engineered nucleic acid each comprises RNA, DNA, ora combination thereof.

12. The complex of any one of embodiments 5 to 11, wherein the firstnucleic acid binding Class 2 Type V CRISPR protein binding sequence andthe second nucleic acid binding Class 2 Type V CRISPR protein bindingsequence are each a Cpf1 protein binding sequence.

13. The complex of embodiment 7 or 11, wherein the first nucleic acidbinding Class 2 Type V CRISPR protein binding sequence, the secondnucleic acid binding Class 2 Type V CRISPR protein binding sequence, thethird nucleic acid binding Class 2 Type 5 CRISPR protein bindingsequence, and the fourth nucleic acid binding Class 2 Type V CRISPRprotein binding sequence are each a Cpf1 protein binding sequence.

14. The complex of any one of embodiments 5, 6, 7, 8, 9, or 10, wherein(i) the nucleic acid sequence 1 further comprises a spacer nucleic acidsequence 1 and the nucleic acid sequence 2 further comprises a spacernucleic acid sequence 2, and (ii) the spacer nucleic acid sequence 1 iscomplementary to a target nucleic acid sequence 1 and the spacer nucleicacid sequence 2 is complementary to a target nucleic acid sequence 2.

15. The complex of embodiment 14, wherein target nucleic acid sequence 1and target nucleic acid sequence 2 are each a nucleic acid sequenceselected from the group consisting of a single-stranded RNA, asingle-stranded DNA, a double-stranded RNA, a double-stranded DNA, asingle-stranded RNA/DNA hybrid, and a double-stranded RNA/DNA hybrid.

16. The complex of any one of embodiments 7, 11, or 13, wherein (i) thenucleic acid sequence 1 further comprises a spacer nucleic acid sequence1, the nucleic acid sequence 2 further comprises a spacer nucleic acidsequence 2, the nucleic acid sequence 3 further comprises a spacernucleic acid sequence 3, and the nucleic acid sequence 4 furthercomprises a spacer nucleic acid sequence 4, and (ii) the spacer nucleicacid sequence 1 is complementary to a target nucleic acid sequence 1,the spacer nucleic acid sequence 2 is complementary to a target nucleicacid sequence 2, the spacer nucleic acid sequence 3 is complementary toa target nucleic acid sequence 3, and the spacer nucleic acid sequence 4is complementary to a target nucleic acid sequence 4.

17. The complex of embodiment 16, wherein the target nucleic acidsequence 1, the target nucleic acid sequence 2, the target nucleic acid3, and the target nucleic acid 4 are each a nucleic acid sequenceselected from the group consisting of a single-stranded RNA, asingle-stranded DNA, a double-stranded RNA, a double-stranded DNA, asingle-stranded RNA/DNA hybrid, and a double-stranded RNA/DNA hybrid.

18. A complex of the two or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 1 to 17, the complexfurther comprising a first Class 2 Type V CRISPR protein bound to thefirst nucleic acid binding Class 2 Type V CRISPR protein bindingsequence, and a second Class 2 Type V CRISPR protein bound to the secondnucleic acid binding Class 2 Type V CRISPR protein binding sequence,wherein the first Class 2 Type V CRISPR protein and the second Class 2Type V CRISPR protein are each selected from the group consisting of aCpf1 protein and a catalytically inactive Cpf1 protein.

19. A complex of the two or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 7, 11, 13, or 16, thecomplex further comprising a first Class 2 Type V CRISPR protein boundto the first nucleic acid binding Class 2 Type V CRISPR protein bindingsequence, a second Class 2 Type V CRISPR protein bound to the secondnucleic acid binding Class 2 Type V CRISPR protein binding sequence, athird Class 2 Type V CRISPR protein bound to the third nucleic acidbinding Class 2 Type V CRISPR protein binding sequence, and a fourthClass 2 Type V CRISPR protein bound to the fourth nucleic acid bindingClass 2 Type V CRISPR protein binding sequence, wherein the first Class2 Type V CRISPR protein, the second Class 2 Type V CRISPR protein, thethird Class 2 Type V CRISPR protein, and the fourth Class 2 Type VCRISPR protein are each selected from the group consisting of a Cpf1protein and a catalytically inactive Cpf1 protein.

20. The complex of embodiment 4, wherein the first nucleic acid bindingClass 2 CRISPR protein binding sequence is a Class 2 Type II CRISPRprotein binding sequence, wherein the first end is a 5′ end and thesecond end is a 3′ end—the repeat nucleic acid sequence 1 has a 5′ endand a 3′ end, wherein the 3′ end of the repeat nucleic acid sequence 1is located 5′ of the 5′ end of the first nucleic acid binding Class 2Type II CRISPR protein binding sequence—and the nucleic acid sequence 1has a 5′ end and a 3′ end, wherein the 3′ end of the nucleic acidsequence 1 is located 5′ of the 5′ end of the repeat nucleic acidsequence 1;—and the second nucleic acid binding Class 2 CRISPR proteinbinding sequence is a Class 2 Type II CRISPR protein binding sequence,wherein the first end is a 5′ end and the second end is a 3′ end—therepeat nucleic acid sequence 1C has a 5′ end and a 3′ end, wherein the3′ end of the repeat nucleic acid sequence 1C is located 5′ of the 5′end of the second nucleic acid binding Class 2 Type II CRISPR proteinbinding sequence—and the nucleic acid sequence 2, having a 5′ end and a3′ end, wherein the 3′ end of the nucleic acid sequence 2 is located 5′of the 5′ end of the repeat nucleic acid sequence 1C.

21. The complex of embodiment 20, wherein the repeat nucleic acidsequence 1 further comprises a linker element nucleic acid sequence 1-1,having a 5′ end and a 3′ end—a repeat nucleic acid sequence 1a, having a5′ end and a 3′ end—a linker element nucleic acid sequence 1-2, having a5′ end and a 3′ end—a repeat nucleic acid sequence 1b, having a 5′ endand a 3′ end—and a linker element nucleic acid sequence 1-3, having a 5′end and a 3′ end, arranged in the following 3′ to 5′ order: the linkerelement nucleic acid sequence 1-1, the repeat nucleic acid sequence 1a,the linker element nucleic acid sequence 1-2, the repeat nucleic acidsequence 1b, and the linker element nucleic acid sequence 1-3;—and therepeat nucleic acid sequence 2 further comprises a linker elementnucleic acid sequence 2-1, having a 5′ end and a 3′ end—a repeat nucleicacid sequence 1aC, having a 5′ end and a 3′ end—a linker element nucleicacid sequence 2-2, having a 5′ end and a 3′ end—a repeat nucleic acidsequence 1bC, having a 5′ end and a 3′ end—and a linker element nucleicacid sequence 2-3, having a 5′ end and a 3′ end, arranged in thefollowing 3′ to 5′ order: the linker element nucleic acid sequence 2-3,the repeat nucleic acid sequence 1bC, the linker element nucleic acidsequence 2-2, the repeat nucleic acid sequence 1aC, and the linkerelement nucleic acid sequence 2-1; wherein the repeat nucleic acidsequence 1 is associated with the repeat nucleic acid sequence 2 throughhydrogen bonding between the repeat nucleic acid sequence 1a and therepeat nucleic acid sequence 1aC and through hydrogen bonding betweenthe repeat nucleic acid sequence 1b and the repeat nucleic acid sequence1bC.

22. The complex of embodiment 21, wherein the repeat nucleic acidsequence 1a further comprises a repeat nucleic acid sequence 1a1, havinga 5′ end and a 3′ end—a bulge nucleic acid sequence 1a1, having a 5′ endand a 3′ end—and a repeat nucleic acid sequence 1a2, having a 5′ end anda 3′ end, arranged in the following 3′ to 5′ order: the repeat nucleicacid sequence 1a1, the bulge nucleic acid sequence 1a1, and the repeatnucleic acid sequence 1a2;—and the repeat nucleic acid sequence 1bfurther comprises a repeat nucleic acid sequence 1b1, having a 5′ endand a 3′ end—a bulge nucleic acid sequence 1b1, having a 5′ end and a 3′end—and a repeat nucleic acid sequence 1b2, having a 5′ end and a 3′end, arranged in the following 3′ to 5′ order: the repeat nucleic acidsequence 1b1, the bulge nucleic acid sequence 1b 1, and the repeatnucleic acid sequence 1b2; and the repeat nucleic acid sequence 1bCfurther comprises a repeat nucleic acid sequence 1b2C, having a 5′ endand a 3′ end—a bulge nucleic acid sequence 2b2, having a 5′ end and a 3′end—and a repeat nucleic acid sequence 1b1C, having a 5′ end and a 3′end, arranged in the following 3′ to 5′ order: the repeat nucleic acidsequence 1b2C, the bulge nucleic acid sequence 2b2, and the repeatnucleic acid sequence 1b1C—and the repeat nucleic acid sequence 1aCfurther comprises a repeat nucleic acid sequence 1a2C, having a 5′ endand a 3′ end—a bulge nucleic acid sequence 2a2, having a 5′ end and a 3′end—and a repeat nucleic acid sequence 1a1C, having a 5′ end and a 3′end, arranged in the following 3′ to 5′ order: the repeat nucleic acidsequence 1a2C, the bulge nucleic acid sequence 2a2, and the repeatnucleic acid sequence 1a1C; wherein the repeat nucleic acid sequence 1is associated with the repeat nucleic acid sequence 2 through hydrogenbonding between the repeat nucleic acid sequence 1a1 and the repeatnucleic acid sequence 1a1C, the repeat nucleic acid sequence 1a2 and therepeat nucleic acid sequence 1a2C, the repeat nucleic acid sequence 1b1and the repeat nucleic acid sequence 1b1C, the repeat nucleic acidsequence 1b2 and the repeat nucleic acid sequence 1b2C.

23. The complex of any one of embodiments 20, 21, or 22, wherein therepeat nucleic acid sequence 1 and the repeat nucleic acid sequence 2comprise a double-stranded nucleic acid binding protein binding site 1and the double-stranded nucleic acid binding protein binding site 1 isformed by hydrogen base-pair bonding between the repeat nucleic acidsequence 1 and the repeat nucleic acid sequence 2.

24. The complex of embodiment 23, wherein the double-stranded nucleicacid binding protein binding site 1 is a Csy4 protein binding site.

25. The complex of any one of embodiments 20 to 24, wherein the firstengineered nucleic acid and the second engineered nucleic acid eachcomprises RNA, DNA, or a combination thereof.

26. The complex of any one of embodiments 20 to 25, wherein the firstnucleic acid binding Class 2 Type II CRISPR protein binding sequence andthe second nucleic acid binding Class 2 Type II CRISPR protein bindingsequence are each a Cas9 protein binding sequence.

27. The complex of any one of embodiments 20 to 26, wherein (i) thenucleic acid sequence 1 further comprises a spacer nucleic acid sequence1 and the nucleic acid sequence 2 further comprises a spacer nucleicacid sequence 2, and (ii) the spacer nucleic acid sequence 1 iscomplementary to a target nucleic acid sequence 1 and the spacer nucleicacid sequence 2 is complementary to a target nucleic acid sequence 2.

28. The complex of embodiment 27, wherein the target nucleic acidsequence 1 and the target nucleic acid sequence 2 are each a nucleicacid sequence selected from the group consisting of a single-strandedRNA, a single-stranded DNA, a double-stranded RNA, a double-strandedDNA, a single-stranded RNA/DNA hybrid, and a double-stranded RNA/DNAhybrid.

29. A complex of the two or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 20 to 28, the complexfurther comprising a first Class 2 Type II CRISPR protein bound to thefirst nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, and a second Class 2 Type II CRISPR protein bound to thesecond nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, wherein the first Class 2 Type II CRISPR protein and thesecond Class 2 Type II CRISPR protein are each selected from the groupconsisting of a Cas9 protein and a catalytically inactive Cas9 protein.

30. A complex of three or more engineered nucleic acid sequences forminga scaffold, comprising: a first engineered nucleic acid comprising, afirst CRISPR element 1 comprising—a first nucleic acid binding Class 2Type II CRISPR protein binding sequence, having a 5′ end and a 3′end—and a repeat nucleic acid sequence 1, having a 5′ end and a 3′ end,wherein the 3′ end of the repeat nucleic acid sequence 1 is located 5′of the 5′ end of the first nucleic acid binding Class 2 Type II CRISPRprotein binding sequence, the repeat nucleic acid sequence 1 furthercomprising, a linker element nucleic acid sequence 1-1, having a 5′ endand a 3′ end—a repeat nucleic acid sequence 1a, having a 5′ end and a 3′end—a linker element nucleic acid sequence 1-2, having a 5′ end and a 3′end—a repeat nucleic acid sequence 1b, having a 5′ end and a 3′ end—anda linker element nucleic acid sequence 1-3, having a 5′ end and a 3′end, arranged in the following 3′ to 5′ order: the linker elementnucleic acid sequence 1-1, the repeat nucleic acid sequence 1a, thelinker element nucleic acid sequence 1-2, the repeat nucleic acidsequence 1b, and the linker element nucleic acid sequence 1-3;—and asecond CRISPR element 1 further comprising a nucleic acid sequence 1,having a 5′ end and a 3′ end, wherein (i) the 3′ end of the nucleic acidsequence 1 is located 5′ of the 5′ end of the repeat nucleic acidsequence 1, and (ii) the nucleic acid sequence 1 comprises a spacernucleic acid sequence 1;—a second engineered nucleic acid comprising,—afirst CRISPR element 2 comprising—a second nucleic acid binding Class 2Type II CRISPR protein binding sequence, having a 5′ end and a 3′end—and a repeat nucleic acid sequence 2, having a 5′ end and a 3′ end,wherein the 3′ end of the repeat nucleic acid sequence 2 is located 5′of the 5′ end of the second nucleic acid binding Class 2 Type II CRISPRprotein binding sequence, the repeat nucleic acid sequence 2 furthercomprising—a linker element nucleic acid sequence 2-3, having a 5′ endand a 3′ end—a repeat nucleic acid sequence 1bC, having a 5′ end and a3′ end—a linker element nucleic acid sequence 2-4, having a 5′ end and a3′ end—a repeat nucleic acid sequence 2a, having a 5′ end and a 3′end—and a linker element nucleic acid sequence 2-5, having a 5′ end anda 3′ end, arranged in the following 3′ to 5′ order: the linker elementnucleic acid sequence 2-3, the repeat nucleic acid sequence 1bC, thelinker element nucleic acid sequence 2-4, the repeat nucleic acidsequence 2a, and the linker element nucleic acid sequence 2-5;—a secondCRISPR element 2 comprising a nucleic acid sequence 2, having a 5′ endand a 3′ end, wherein (i) the 3′ end of the nucleic acid sequence 1 islocated 5′ of the 5′ end of the repeat nucleic acid sequence 2, and (ii)the nucleic acid sequence 2 comprises a spacer nucleic acid sequence2;—and a third engineered nucleic acid comprising, a first CRISPRelement 3 comprising a third nucleic acid binding Class 2 Type II CRISPRprotein binding sequence, having a 5′ end and a 3′ end—and a repeatnucleic acid sequence 3, having a 5′ end and a 3′ end, wherein the 3′end of the repeat nucleic acid sequence 3 is located 5′ of the 5′ end ofthe third nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, the repeat nucleic acid sequence 3 further comprising a linkerelement nucleic acid sequence 3-1, having a 5′ end and a 3′ end—a repeatnucleic acid sequence 2aC, having a 5′ end and a 3′ end—a linker elementnucleic acid sequence 3-2, having a 5′ end and a 3′ end—a repeat nucleicacid sequence 1aC-1, having a 5′ end and a 3′ end—and a linker elementnucleic acid sequence 3-3, having a 5′ end and a 3′ end, arranged in thefollowing 3′ to 5′ order: the linker element nucleic acid sequence 3-1,the repeat nucleic acid sequence 2aC, the linker element nucleic acidsequence 3-2, the repeat nucleic acid sequence 1aC-1, and the linkerelement nucleic acid sequence 3-3; a second CRISPR element 3 comprisinga nucleic acid sequence 3, having a 5′ end and a 3′ end, wherein (i) the3′ end of the nucleic acid sequence 3 is located 5′ of the 5′ end of therepeat nucleic acid sequence 3, and (ii) the nucleic acid sequence 3comprises a spacer nucleic acid sequence 3; wherein repeat nucleic acidsequence 1a is associated with the repeat nucleic acid sequence 1aC-1through hydrogen bonding between the repeat nucleic acid sequence 1a andthe repeat nucleic acid sequence 1aC-1, the repeat nucleic acid sequence1b is associated with the repeat nucleic acid sequence 1bC throughhydrogen bonding between the repeat nucleic acid sequence 1b and therepeat nucleic acid sequence 1bC, and the repeat nucleic acid sequence2a is associated with the repeat nucleic acid sequence 2aC throughhydrogen bonding between the repeat nucleic acid sequence 2a and therepeat nucleic acid sequence 2aC.

31. The complex of embodiment 30, wherein the repeat nucleic acidsequence 1 and the repeat nucleic acid sequence 2 comprise adouble-stranded nucleic acid binding protein binding site 1 and thedouble-stranded nucleic acid binding protein binding site 1 is formed byhydrogen base-pair bonding between the repeat nucleic acid sequence 1and the repeat nucleic acid sequence 2.

32. The complex of embodiment 31, wherein the double-stranded nucleicacid binding protein binding site 1 is a Csy4 protein binding site.

33. The complex of any one of embodiments 30, 31, or 32, wherein thefirst engineered nucleic acid, the second engineered nucleic acid, andthe third engineered nucleic acid each comprises RNA, DNA, or acombination thereof.

34. The complex of any one of embodiments 30 to 33, wherein the firstnucleic acid binding Class 2 Type II CRISPR protein binding sequence,the second nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, and the third nucleic acid binding Class 2 Type II CRISPRprotein binding sequence are each a Cas9 protein binding sequence.

35. The complex of any one of embodiments 30 to 34, wherein (i) thenucleic acid sequence 1 further comprises a spacer nucleic acid sequence1, the nucleic acid sequence 2 further comprises a spacer nucleic acidsequence 2, and the nucleic acid sequence 3 further comprises a spacernucleic acid sequence 3, and (ii) the spacer nucleic acid sequence 1 iscomplementary to a target nucleic acid sequence 1, the spacer nucleicacid sequence 2 is complementary to a target nucleic acid sequence 2,and the spacer nucleic acid sequence 3 is complementary to a targetnucleic acid sequence 3.

36. The complex of embodiment 35, wherein the target nucleic acidsequence 1, the target nucleic acid sequence 2, and the target nucleicacid sequence 3 are each a nucleic acid sequence selected from the groupconsisting of a single-stranded RNA, a single-stranded DNA, adouble-stranded RNA, a double-stranded DNA, a single-stranded RNA/DNAhybrid, and a double-stranded RNA/DNA hybrid.

37. A complex of the three or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 30 to 36, the complexfurther comprising a first Class 2 Type II CRISPR protein bound to thefirst nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, a second Class 2 Type II CRISPR protein bound to the secondnucleic acid binding Class 2 Type II CRISPR protein binding sequence,and a third Class 2 Type II CRISPR protein bound to the third nucleicacid binding Class 2 Type II CRISPR protein binding sequence, whereinthe first Class 2 Type II CRISPR protein, the second Class 2 Type IICRISPR protein, and the third Class 2 Type II CRISPR protein are eachselected from the group consisting of a Cas9 protein and a catalyticallyinactive Cas9 protein.

38. A complex of two or more engineered nucleic acid sequences forming ascaffold, comprising: an engineered concatenated nucleic acid 1 having a5′ end and a 3′ end comprising a first concatenate element 1 comprisinga first nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, having a 5′ end and a 3′ end—a second concatenate element 1comprising a repeat nucleic acid sequence A1 having a 5′ end and a 3′end wherein the first nucleic acid binding Class 2 Type II CRISPRprotein binding sequence is located 3′ of the 3′ end of the repeatnucleic acid sequence A1—a first concatenate element 2 comprising asecond nucleic acid binding Class 2 Type II CRISPR protein bindingsequence, having a 5′ end and a 3′ end—a second concatenate element 2comprising a repeat nucleic acid sequence A2, having a 5′ end and a 3′end, wherein the second nucleic acid binding Class 2 Type II CRISPRprotein binding sequence is located 3′ of the 3′ end of the repeatnucleic acid sequence A2, wherein the 5′ end of the first concatenateelement 1 is covalently bound to the 3′ end of the first concatenateelement 2 to form the engineered concatenated nucleic acid 1;—a thirdconcatenate element 1 having a 5′ end and a 3′ end comprising a repeatnucleic acid sequence A1C having a 5′ end and a 3′ end and a nucleicacid sequence 1 having a 5′ end and a 3′ end, wherein the nucleic acidsequence 1 is located 5′ of the 5′ end of the repeat nucleic acidsequence A1C, wherein (i) the repeat nucleic acid sequence A1C iscomplementary to the repeat nucleic acid sequence A1, (ii) the repeatnucleic acid sequence A1C is associated with the repeat nucleic acidsequence A1 through hydrogen bonding between the repeat nucleic acidsequence A1C and the repeat nucleic acid sequence A1;—and a thirdconcatenate element 2 having a 5′ end and a 3′ end comprising a repeatnucleic acid sequence A2C having a 5′ end and a 3′ end and a nucleicacid sequence 2 having a 5′ end and a 3′ end, wherein the nucleic acidsequence 2 is located 5′ of the 5′ end of the repeat nucleic acidsequence A2C, wherein (i) the repeat nucleic acid sequence A2C iscomplementary to the repeat nucleic acid sequence A2, (ii) the repeatnucleic acid sequence A2C is associated to the repeat nucleic acidsequence A2, and (iii) the repeat nucleic acid sequence A2C isassociated with the repeat nucleic acid sequence A2 through hydrogenbonding between the repeat nucleic acid sequence A2C and the repeatnucleic acid sequence A2.

39. The complex of embodiment 38, wherein the repeat nucleic acidsequence A1 further comprises a linker element nucleic acid sequenceA1-1 having a 5′ end and a 3′ end, the 3′ end of the linker elementnucleic acid sequence A1-1 located 5′ of the 5′ end of the first nucleicacid binding Class 2 Type II CRISPR protein binding sequence, the linkerelement nucleic acid sequence A1-1 comprising, a repeat nucleic acidsequence A1-1 having a 5′ end and a 3′ end, and a bulge nucleic acidsequence A1-1, having a 5′ end and a 3′ end, the 3′ end of the bulgenucleic acid sequence A1-1 adjacent the 5′ end of the repeat nucleicacid sequence A1-1—and a linker element nucleic acid sequence A1-2,having a 5′ end and a 3′ end, comprising, a repeat nucleic acid sequenceA1-2, having a 5′ end and a 3′ end, the 3′ end of the linker elementnucleic acid sequence A1-2 located 5′ of the 5′ end of the linkerelement nucleic acid A1-1;—the repeat nucleic acid sequence A2 furthercomprises a linker element nucleic acid sequence A2-1 having a 5′ endand a 3′ end, the 3′ end of the linker element nucleic acid sequenceA2-1 located 5′ of the 5′ end of the second nucleic acid binding Class 2Type II CRISPR protein binding sequence, the linker element nucleic acidsequence A2-1 comprising, a repeat nucleic acid sequence A2-1 having a5′ end and a 3′ end, and a bulge nucleic acid sequence A2-1, having a 5′end and a 3′ end, the 3′ end of the bulge nucleic acid sequence A2-1adjacent the 5′ end of the repeat nucleic acid sequence A2-1—and alinker element nucleic acid sequence A2-2, having a 5′ end and a 3′ end,comprising, a repeat nucleic acid sequence A2-2, having a 5′ end and a3′ end, the 3′ end of the linker element nucleic acid sequence A2-2located 5′ of the 5′ end of the linker element nucleic acid A2-1;—thethird concatenate element 1 wherein the repeat nucleic acid sequence A1Cfurther comprises a linker element nucleic acid sequence A1-1Ccomprising a repeat nucleic acid sequence A1-1C having a 5′ end and a 3′end, the 5′ end of the repeat nucleic acid sequence A1-1C located 3′ ofthe 3′ end of the nucleic acid sequence 1, and a bulge nucleic acidsequence A1-1C, having a 5′ end and a 3′ end, the 5′ end of the bulgenucleic acid sequence A1-1C located 3′ of the 3′ end of the repeatnucleic acid sequence A1-1C, wherein (i) the repeat nucleic acidsequence A1-1C is complementary to the repeat nucleic acid sequenceA1-1, and (ii) the repeat nucleic acid sequence A1-1C is associated withthe repeat nucleic acid sequence A1-1 through hydrogen bonding betweenthe repeat nucleic acid sequence A1-1C and the repeat nucleic acidsequence A1-1—and a linker element nucleic acid sequence A1-2C having a5′ end and a 3′ end, comprising a repeat nucleic acid sequence A1-2C,having a 5′ end and a 3′ end, the 5′ end of the linker element nucleicacid sequence A1-2C located 3′ of the 3′ end of the linker elementnucleic acid sequence A1-1C, wherein (i) the repeat nucleic acidsequence A1-2C is complementary to the repeat nucleic acid sequenceA1-2, and (ii) the repeat nucleic acid sequence A1-2C is associated withthe repeat nucleic acid sequence A1-2 through hydrogen bonding betweenthe repeat nucleic acid sequence A1-2C and the repeat nucleic acidsequence A1-2;—and the repeat nucleic acid sequence A2C furthercomprises a linker element nucleic acid sequence A2-1C comprising arepeat nucleic acid sequence A2-1C having a 5′ end and a 3′ end, the 5′end of the repeat nucleic acid sequence A2-1C located 3′ of the 3′ endof the nucleic acid sequence 2, and a bulge nucleic acid sequence A2-1C,having a 5′ end and a 3′ end, the 5′ end of the bulge nucleic acidsequence A2-1C located 3′ of the 3′ end of the repeat nucleic acidsequence A2-1C, wherein (i) the repeat nucleic acid sequence A2-1C iscomplementary to the repeat nucleic acid sequence A2-1, and (ii) therepeat nucleic acid sequence A2-1C is associated with the repeat nucleicacid sequence A2-1 through hydrogen bonding between the repeat nucleicacid sequence A2-1C and the repeat nucleic acid sequence A2-1—and alinker element nucleic acid sequence A2-2C having a 5′ end and a 3′ end,comprising a repeat nucleic acid sequence A2-2C, having a 5′ end and a3′ end, the 5′ end of the linker element nucleic acid sequence A2-2Clocated 3′ of the 3′ end of the linker element nucleic acid sequenceA2-1C, wherein (i) the repeat nucleic acid sequence A2-2C iscomplementary to the repeat nucleic acid sequence A2-2, and (ii) therepeat nucleic acid sequence A2-2C is associated with the repeat nucleicacid sequence A2-2 through hydrogen bonding between the repeat nucleicacid sequence A2-2C and the repeat nucleic acid sequence A2-2.

40. The complex of embodiment 38 or 39, wherein the repeat nucleic acidsequence A1 and the repeat nucleic acid sequence A1C further comprise adouble-stranded nucleic acid binding protein binding site 1 and thedouble-stranded nucleic acid binding protein binding site 1 is formed byhydrogen base-pair bonding between the repeat nucleic acid sequence A1and the repeat nucleic acid sequence A1C.

41. The complex of any one of embodiments 38 to 40, wherein the repeatnucleic acid sequence A2 and the repeat nucleic acid sequence A2Cfurther comprise a double-stranded nucleic acid binding protein bindingsite 2 and the double-stranded nucleic acid binding protein binding site2 is formed by hydrogen base-pair bonding between the repeat nucleicacid sequence A2 and the repeat nucleic acid sequence A2C.

42. The complex of embodiment 40 or 41, wherein the double-strandednucleic acid binding protein binding site 1 is a Csy4 protein bindingsite.

43. The complex of any one of 38 to 42, wherein the engineeredconcatenated nucleic acid 1, the third concatenate element 1, and thethird concatenate element 2 each comprises RNA, DNA, or a combinationthereof.

44. The complex of any one of 38 to 43, wherein the first nucleic acidbinding Class 2 Type II CRISPR protein binding sequence and the secondnucleic acid binding Class 2 Type II CRISPR protein binding sequence areeach a Cas9 protein binding sequence.

45. The complex of any one of 38 to 44, wherein (i) the nucleic acidsequence 1 further comprises a spacer nucleic acid sequence 1 and thenucleic acid sequence 2 further comprises a spacer nucleic acid sequence2, and (ii) the spacer nucleic acid sequence 1 is complementary to atarget nucleic acid sequence 1 and the spacer nucleic acid sequence 2 iscomplementary to a target nucleic acid sequence 2.

46. The complex of embodiment 45, wherein the target nucleic acidsequence 1 and the target nucleic acid sequence 2 are each a nucleicacid sequence selected from the group consisting of a single-strandedRNA, a single-stranded DNA, a double-stranded RNA, a double-strandedDNA, a single-stranded RNA/DNA hybrid, and a double-stranded RNA/DNAhybrid.

47. A complex of the two or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 38 to 46 the complexfurther comprising a first Class 2 Type II CRISPR protein bound to thefirst nucleic acid binding Class 2 Type II CRISPR protein bindingsequence and a second Class 2 Type II CRISPR protein bound to the secondnucleic acid binding Class 2 Type II CRISPR protein binding sequence,wherein the first Class 2 Type II CRISPR protein and the second Class 2Type II CRISPR protein are each selected from the group consisting of aCas9 protein and a catalytically inactive Cas9 protein.

48. A complex of two or more engineered nucleic acid sequences forming ascaffold, comprising: an engineered concatenated split-nexus nucleicacid 1, having a 5′ end and a 3′ end, comprising a first split-nexuselement 1, having a 5′ end and a 3′ end, comprising a first nucleic acidbinding Class 2 Type II CRISPR protein binding sequence and asplit-nexus stem element nucleic acid sequence 1-1 having a 5′ end and a3′ end wherein the first nucleic acid binding Class 2 Type II CRISPRprotein binding sequence is located 3′ to the 3′ end of the split-nexusstem element nucleic acid sequence 1-1—and a first split-nexus element 2having a 5′ end and a 3′ end, comprising a second nucleic acid bindingClass 2 Type II CRISPR protein binding sequence and a split-nexus stemelement nucleic acid sequence 2-1 having a 5′ end and a 3′ end whereinthe second nucleic acid binding Class 2 Type II CRISPR protein bindingsequence is located 3′ to the 3′ end of the split-nexus stem elementnucleic acid sequence 2-1,—and an auxiliary polynucleotide 1-1 having a5′ end and a 3′ end, wherein the 5′ end of the first split-nexus element1 is covalently bound to the 3′ end of the auxiliary polynucleotide 1-1,and the 5′ end of the auxiliary polynucleotide 1-1 is covalently boundto the 3′ end of the first split-nexus element 2 to form theconcatenated split-nexus element;—a second split-nexus element 1, havinga 5′ end and a 3′ end, comprising a nucleic acid sequence 1 having a 5′end and a 3′ end and a first stem element nucleic acid sequence 1-1having a 5′ end and a 3′ end, wherein the 3′ end of the nucleic acidsequence 1 is covalently bound to the 5′ end of the first stem elementnucleic acid sequence 1-1—a loop element nucleic acid sequence 1 havinga 5′ end and a 3′ end, wherein the 3′ end of the first stem elementnucleic acid sequence 1-1 is covalently bound to the 5′ end of the loopelement nucleic acid sequence 1—a first stem element nucleic acidsequence 1-2 having a 5′ end and a 3′ end, wherein the 3′ end of theloop element nucleic acid sequence 1 is covalently bound to the 5′ endof the first stem element nucleic acid sequence 1-2—a connective nucleicacid sequence 1 having a 5′ end and a 3′ end, wherein the 3′ end of thefirst stem element nucleic acid sequence 1-2 is covalently bound to the5′ end of the connective nucleic acid sequence 1—and a split-nexus stemelement nucleic acid sequence 1-2, wherein the 3′ end of the connectivenucleic acid sequence 1 is covalently bound to the 5′ end of thesplit-nexus stem element nucleic acid sequence 1-2, wherein (i) thefirst stem element nucleic acid sequence 1-1 and the first stem elementnucleic acid sequence 1-2 form a first stem element 1 by hydrogenbase-pair bonding between the first stem element nucleic acid sequence1-1 and the first stem element nucleic acid sequence 1-2, and (ii) thesplit-nexus stem element nucleic acid sequence 1-1 and the split-nexusstem element nucleic acid sequence 1-2 form a split-nexus stem element 1by hydrogen base-pair bonding between the split-nexus stem elementnucleic acid sequence 1-1 and the split-nexus stem element nucleic acidsequence 1-2;—and a second split-nexus element 2, having a 5′ end and a3′ end, comprising a nucleic acid sequence 2 having a 5′ end and a 3′end and a first stem element nucleic acid sequence 2-1 having a 5′ endand a 3′ end, wherein the 3′ end of the nucleic acid sequence 2 iscovalently bound to the 5′ end of the first stem element nucleic acidsequence 2-1—a loop element nucleic acid sequence 2 having a 5′ end anda 3′ end, wherein the 3′ end of the first stem element nucleic acidsequence 2-1 is covalently bound to the 5′ end of the loop elementnucleic acid sequence 2—a first stem element nucleic acid sequence 2-2having a 5′ end and a 3′ end, wherein the 3′ end of the loop elementnucleic acid sequence 2 is covalently bound to the 5′ end of the firststem element nucleic acid sequence 2-2—a connective nucleic acidsequence 2 having a 5′ end and a 3′ end, wherein the 3′ end of the firststem element nucleic acid sequence 2-2 is covalently bound to the 5′ endof the connective nucleic acid sequence 2—and a split-nexus stem elementnucleic acid sequence 2-2, wherein the 3′ end of the connective nucleicacid sequence 1 is covalently bound to the 5′ end of the split-nexusstem element nucleic acid sequence 2-2, wherein (i) the first stemelement nucleic acid sequence 2-1 and the first stem element nucleicacid sequence 2-2 form a first stem element 2 by hydrogen base-pairbonding between the first stem element nucleic acid sequence 2-1 and thefirst stem element nucleic acid sequence 2-2, and (ii) the split-nexusstem element nucleic acid sequence 2-1 and the split-nexus stem elementnucleic acid sequence 2-2 form a split-nexus stem element 2 by hydrogenbase-pair bonding between the split-nexus stem element nucleic acidsequence 2-1 and the split-nexus stem element nucleic acid sequence 2-2.

49. The complex of embodiment 48, wherein the first stem element 1further comprises in a 5′ to 3′ direction a lower stem element nucleicacid sequence 1-1, a bulge element nucleic acid sequence 1-1, an upperstem element nucleic acid sequence 1-1, the loop element nucleic acidsequence 1, an upper stem element nucleic acid sequence 1-2, a bulgeelement nucleic acid sequence 1-2, and a lower stem element nucleic acidsequence 1-2, wherein the upper stem element nucleic acid sequence 1-1and the upper stem element nucleic acid sequence 1-2 form an upper stemelement 1 by hydrogen base-pair bonding between the upper stem elementnucleotide sequence 1-1 and the upper stem element nucleotide sequence1-2, and the lower stem element nucleic acid sequence 1-1 and the lowerstem element nucleic acid sequence 1-2 form a lower stem element 1 byhydrogen base-pair bonding between the lower stem element nucleic acidsequence 1-1 and the lower stem element nucleotide sequence 1-2.

50. The complex of embodiment 48 or 49, wherein the first stem element 2further comprises in a 5′ to 3′ direction a lower stem element nucleicacid sequence 2-1, a bulge element nucleic acid sequence 2-1, an upperstem element nucleic acid sequence 2-1, the loop element nucleic acidsequence 2, an upper stem element nucleic acid sequence 2-2, a bulgeelement nucleic acid sequence 2-2, and a lower stem element nucleic acidsequence 2-2, wherein the upper stem element nucleic acid sequence 2-1and the upper stem element nucleic acid sequence 2-2 form an upper stemelement 2 by hydrogen base-pair bonding between the upper stem elementnucleotide sequence 2-1 and the upper stem element nucleotide sequence2-2, and the lower stem element nucleic acid sequence 2-1 and the lowerstem element nucleic acid sequence 2-2 form a lower stem element 2 byhydrogen base-pair bonding between the lower stem element nucleic acidsequence 2-1 and the lower stem element nucleotide sequence 2-2.

51. The complex of any one of embodiments 48 to 50, wherein the secondsplit-nexus element 1 further comprises an auxiliary polynucleotide 1-2,having a 5′ and a 3′ end, wherein 5′ end of the auxiliary polynucleotide1-2 is 3′ of the 3′ end of the split-nexus stem element nucleic acidsequence 1-2, wherein the auxiliary polynucleotide 1-2 is associatedwith the auxiliary polynucleotide 1-1 through hydrogen base-pairbonding.

52. The complex of embodiment 51, wherein the auxiliary polynucleotide1-1 and the auxiliary polynucleotide 1-2 further comprise adouble-stranded nucleic acid binding protein binding site 1 and thedouble-stranded nucleic acid binding protein binding site 1 is formed byhydrogen base-pair bonding between the auxiliary polynucleotide 1-1 andthe auxiliary polynucleotide 1-2.

53. The complex of embodiment 52, wherein the double-stranded nucleicacid binding protein binding site 1 is a Csy4 protein binding site 1.

54. The complex of any one of embodiments 48 to 53, wherein the secondsplit-nexus element 2 further comprises an auxiliary polynucleotide 2-2,having a 5′ and a 3′ end, wherein 5′ end of the auxiliary polynucleotide2-2 is 3′ of the 3′ end of the split-nexus stem element nucleic acidsequence 2-2—and the first split-nexus element 2 further comprises anauxiliary polynucleotide 2-1 having a 5′ end and a 3′ end, wherein (i)the 5′ end of the first split-nexus element 2 is covalently bound to the3′ end of the auxiliary polynucleotide 2-1, and (ii) the auxiliarypolynucleotide 2-2 is associated with the auxiliary polynucleotide 2-1through hydrogen base-pair bonding.

55. The complex of embodiment 54, wherein the auxiliary polynucleotide2-1 and the auxiliary polynucleotide 2-2 further comprise adouble-stranded nucleic acid binding protein binding site 2 and thedouble-stranded nucleic acid binding protein binding site 2 is formed byhydrogen base-pair bonding between the auxiliary polynucleotide 2-1 andthe auxiliary polynucleotide 2-2.

56. The complex of embodiment 55, wherein the double-stranded nucleicacid binding protein binding site 2 is a Csy4 protein binding site 2.

57. The complex of any one of 48 to 56, wherein the engineeredconcatenated split-nexus nucleic acid 1, the third concatenate element1, and the third concatenate element 2 each comprises RNA, DNA, or acombination thereof.

58. The complex of any one of 48 to 57, wherein the first nucleic acidbinding Class 2 Type II CRISPR protein binding sequence and the secondnucleic acid binding Class 2 Type II CRISPR protein binding sequence areeach a Cas9 protein binding sequence.

59. The complex of any one of 48 to 58, wherein (i) the nucleic acidsequence 1 further comprises a spacer nucleic acid sequence 1 and thenucleic acid sequence 2 further comprises a spacer nucleic acid sequence2, and (ii) the spacer nucleic acid sequence 1 is complementary to atarget nucleic acid sequence 1 and the spacer nucleic acid sequence 2 iscomplementary to a target nucleic acid sequence 2.

60. The complex of embodiment 59, wherein the target nucleic acidsequence 1 and the target nucleic acid sequence 2 are each a nucleicacid sequence selected from the group consisting of a single-strandedRNA, a single-stranded DNA, a double-stranded RNA, a double-strandedDNA, a single-stranded RNA/DNA hybrid, and a double-stranded RNA/DNAhybrid.

61. A complex of the two or more engineered nucleic acid sequencesforming the scaffold of any one of embodiments 48 to 60, the complexfurther comprising a first Class 2 Type II CRISPR protein bound to thefirst nucleic acid binding Class 2 Type II CRISPR protein bindingsequence and a second Class 2 Type II CRISPR protein bound to the secondnucleic acid binding Class 2 Type II CRISPR protein binding sequence,wherein the first Class 2 Type II CRISPR protein and the second Class 2Type II CRISPR protein are each selected from the group consisting of aCas9 protein and a catalytically inactive Cas9 protein.

62. An engineered nucleic acid scaffold, comprising; a first engineerednucleic acid comprising—a first element 1 comprising a first nucleicacid binding Class 2 Type II CRISPR protein binding sequence, having a5′ end and a 3′ end—and a second element 1 comprising a repeat nucleicacid sequence 1 having a 5′ end and a 3′ end, wherein the 3′ end of therepeat nucleic acid sequence 1 is located 5′ of the 5′ end of the firstnucleic acid binding Class 2 Type II CRISPR protein binding sequence,the repeat nucleic acid sequence 1 further comprising a linker elementnucleic acid sequence 1-1, having a 5′ end and a 3′ end—a repeat nucleicacid sequence 1a, having a 5′ end and a 3′ end—a linker element nucleicacid sequence 1-2, having a 5′ end and a 3′ end—a repeat nucleic acidsequence 1b, having a 5′ end and a 3′ end—and a linker element nucleicacid sequence 1-3, having a 5′ end and a 3′ end, arranged in thefollowing 3′ to 5′ order: the linker element nucleic acid sequence 1-1,the repeat nucleic acid sequence 1a, the linker element nucleic acidsequence 1-2, the repeat nucleic acid sequence 1b, and the linkerelement nucleic acid sequence 1-3; wherein no nucleic acid sequencewithin the repeat nucleic acid sequence 1 associates with any nucleicacid sequence within the repeat nucleic acid sequence 1 to form a stemelement through hydrogen bonding capable of binding to a Class 2 Type IICRISPR-Cas protein.

63. The engineered nucleic acid scaffold of embodiment 62, furthercomprising a third element 1 comprising a nucleic acid sequence 1,having a 5′ end and a 3′ end, wherein (i) the 3′ end of the nucleic acidsequence 1 is covalently attached to the 5′ end of the repeat nucleicacid sequence 1, and (ii) the nucleic acid sequence 1 comprises a spacernucleic acid sequence 1.

64. The engineered nucleic acid scaffold of embodiment 62 or 63, furthercomprising: a second engineered nucleic acid comprising—a first element2 comprising a second nucleic acid binding Class 2 Type II CRISPRprotein binding sequence, having a 5′ end and a 3′ end—and a secondelement 2 comprising the repeat nucleic acid sequence 1C has a 5′ endand a 3′ end, wherein the 3′ end of the repeat nucleic acid sequence 1Cis located 5′ of the 5′ end of the second nucleic acid binding Class 2Type II CRISPR protein binding sequence, the repeat nucleic acidsequence 2 further comprising, a linker element nucleic acid sequence2-1, having a 5′ end and a 3′ end—a repeat nucleic acid sequence 1bC,having a 5′ end and a 3′ end—a linker element nucleic acid sequence 2-2,having a 5′ end and a 3′ end—a repeat nucleic acid sequence 1aC, havinga 5′ end and a 3′ end—and a linker element nucleic acid sequence 2-3,having a 5′ end and a 3′ end, arranged in the following 3′ to 5′ order:the linker element nucleic acid sequence 2-3, the repeat nucleic acidsequence 1bC, the linker element nucleic acid sequence 2-2, the repeatnucleic acid sequence 1aC, and the linker element nucleic acid sequence2-1; wherein the repeat nucleic acid sequence 1 is associated with therepeat nucleic acid sequence 2 through hydrogen bonding between therepeat nucleic acid sequence 1a and the repeat nucleic acid sequence 1aCand through hydrogen bonding between the repeat nucleic acid sequence 1band the repeat nucleic acid sequence 1bC.

65. The engineered nucleic acid scaffold of embodiment 64, furthercomprising, a third element 2 comprises a nucleic acid sequence 2,having a 5′ end and a 3′ end, wherein (i) the 3′ end of the nucleic acidsequence 2 is covalently attached to the 5′ end of the repeat nucleicacid sequence 1C, and (ii) the nucleic acid sequence 2 comprises aspacer nucleic acid sequence 2.

Although preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. From theabove description and the following Examples, one skilled in the art canascertain essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make changes,substitutions, variations, and modifications of the invention to adaptit to various usages and conditions. Such changes, substitutions,variations, and modifications are also intended to fall within the scopeof the present disclosure.

Experimental

Aspects of the present invention are illustrated in the followingExamples. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, concentrations, percent changes, and thelike) but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, temperature is in degrees Centigradeand pressure is at or near atmospheric. It should be understood thatthese Examples are given by way of illustration only and are notintended to limit the scope of what the inventors regard as variousaspects of the present invention.

EXAMPLE 1 In Silico Design of NASC Polynucleotide Components

This Example provides a description of the design of NASC polynucleotidecomponents for a number of embodiments of the NASCs described herein.

Table 9 sets forth a correlation between NASC polynucleotide componentsand structures illustrated in the figures. The column “Assoc. Casprotein” lists the Cas proteins with which the NASC polynucleotidecomponent can be used. Unless otherwise indicated, the Cas9 protein is aS. pyogenes Cas9 protein (S. pyogenes Cas9 protein, SEQ ID NO. 100 or S.pyogenes dCas9 protein (SEQ ID NO. 101)). The Cpf1 protein is anAcidaminococcus sp. Cpf1 protein (dCpf SEQ ID NO. 105) unless otherwiseindicated.

Sequences that hybridize between polynucleotide components areunderlined. Nucleic acid target binding sequences are indicated by aseries of twenty Ns, wherein N is any nucleotide. A nucleic acid targetbinding sequence can be engineered by one of ordinary skill in the art.

TABLE 9 Examples of NASC Polynucleotide Component Sequences NASCcomponent Assoc. FIG. generic Cas FIG. indicator designation proteinSequence SEQ ID NO. 4A 1-1, 1-2, NASC-PC1 Cpf1 CUCCGGCGAUGUCACACSEQ ID NO. 1-3 CGAACUGAUAAUUUCUA 64 CUCUUGUAGAUNNNNNN NNNNNNNNNNNNNN 4A2-3, 2-2, NASC-PC2 Cpf1 UCGGUGUGACAUCGCCG SEQ ID NO. 2-1GAGUUGAUAAAUUUCUA 65 CUCUUGUAGAUNNNNNN NNNNNNNNNNNNNN 4B 1-1 NASC-PC1-Cpf1 CUCCGGCGAUGUCACAC SEQ ID NO. 1 CGAACUGAUAAUUUCUA 66 C 4B 1-3NASC-PC1- Cpf1 GUAGAUNNNNNNNNNN SEQ ID NO. 2 NNNNNNNNNN 67 4B 2-1NASC-PC2- Cpf1 UCGGUGUGACAUCGCCG SEQ ID NO. 1 GAGUUGAUAAAUUUGU 68 AG 4B2-3 NASC-PC2- Cpf1 CUACAUNNNNNNNNNNN SEQ ID NO. 2 NNNNNNNNN 69 4C1-1, 1-3, NASC-PC1 Cpf1 AAUUUCUACUCUUGUAG SEQ ID NO. 1-2AUNNNNNNNNNNNNNN 70 NNNNNNACUGAUCUCCG GCGAUGUCACACCGA 4C 2-2, 2-3,NASC-PC2 Cpf1 AAUUUCUACUCUUGUAG SEQ ID NO. 2-2 AUNNNNNNNNNNNNNN 71NNNNNNUUGAUAUCGGU GUGACAUCGCCGGAG 4M 1-1, 1-2, NASC-PC1 Cpf1CUCCGGCGAUGUCACAC SEQ ID NO. 1-3, 1-4 CGAACUGAUAAUUUCUA 72CUCUUGUAGAUNNNNNN NNNNNNNNNNNNNNUU GAUAGUCUAAGGCAGCU AGGGUCU 4M 2-1,2-2,NASC-PC2 Cpf1 UCGGUGUGACAUCGCCG SEQ ID NO. 2-3 GAGUUGAUAAAUUUCUA 73CUCUUGUAGAUNNNNNN NNNNNNNNNNNNNN 4M 3-2, 3-3, NASC-PC3 Cpf1AAUUUCUACUCUUGUAG SEQ ID NO. 3-1 AUNNNNNNNNNNNNNN 74 NNNNNNUACCAAAGACCCUAGCUGCCUUAGAC 5D 507-500 NASC-PC1 Cpf1 CUCCGGCGAUGUCACAC SEQ ID NO. ICGAACUGAUGUCUAAGG 75 CAGCUAGGGUCUUUGAU AAAUUUCUACUCUUGUAGAUNNNNNNNNNNNNN NNNNNNN 5D 508-515 NASC-PC2 Cpf1 UGCGAACCACUGUGAGCSEQ ID NO. II CAGUACCAAUCGGUGUG 76 ACAUCGCCGGAGUUGAU AAAUUUCUACUCUUGUAGAUNNNNNNNNNNNNN NNNNNNN 5F VIII NASC-PC1- Cpf1 CUCCGGCGAUGUCACACSEQ ID NO. 507-503 1 CGAACUGAUGUCUAAGG 77 CAGCUAGGGUCUUUGAU AAAUUUCUAC5F VIII NASC-PC1- Cpf1 GUAGAUNNNNNNNNNN SEQ ID NO. 502-500 2 NNNNNNNNNN78 5F V NASC-PC2- Cpf1 UGCGAACCACUGUGAGC SEQ ID NO. 508-512 1CAGUACCAAUCGGUGUG 79 ACAUCGCCGGAGUUGAU AAAUUUGUAG 5F V NASC-PC2- Cpf1CUACAUNNNNNNNNNNN SEQ ID NO. 513-515 2 NNNNNNNNN 80 6D 611-601 NASC-PC1Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUUUAGUCCCUA 81 AUUAAAUUUCUUGAAAUUGGUAUAUAAGGAGG GACUACAACAAAGAGUU UGCGGGACUCUGCGGGG UUACAAUCCCCUAAAACCGCUUUUAAAAUUCAAA UAAAUUUUGCUUU 6D 612-622 NASC-PC2 Cas9NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUGUAGUCCCUC 82 CUUAUAUACCAAGAAAAAGAAAUUUAAUUAGGG ACUAAAACAAAGAGUUU GCGGGACUCUGCGGGGU UACAAUCCCCUAAAACCGCUUUUAAAAUUCAAAU AAAUUUUGCUUU 6G 611-601 NASC-PC1 Cas9 NNNNNNNNNNNNNNNNSEQ ID NO. NNNNGUCUCAGAGCUAU 83 GCAGUCCUGGACAACUG CCGAACCUCAUGAGAAUCCAAGUAUGUGUAAGGC UAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAG UCGGUGCUU 6G622-612 NASC-PC2 Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGCACAUGAGGAUU 84CUCAUGAGGGACGGCAG AAGAACAGGACUGCAUA GCAAGUUGAGAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUU 6I IV NASC-PC1 Cas9 NNNNNNNNNNNNNNNNSEQ ID NO. NNNNGUUUUAGAGCUAU 85 GCUGUUUUGGAAAGGUC AUGUCCUUCAAAGUUGUAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUG GCACCGAGUCGGUGCUU 6I V NA SC-PC2Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUAAUAGAAUCGU 86 GCUGAAAAGGAAACAAAACAGCAUAGCAAGUUAA AAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUG GCACCGAGUCGGUGCUU6I VI NASC-PC3 Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUACAGAUGAAG 87GACAUGACCGAAACUUU UCAGCACGAUAAGUUAU UAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU 7B 725-722 NASC-PC1 Cas9 NNNNNNNNNNNNNNNN SEQ ID NO.NNNNGUUUUAGUCCCUA 88 AUUAAAUUUCUU 7B 721-718 NASC-PC2 Cas9NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUGUAGUCCCUC 89 CUUAUAUACCAA 7B 717-700NASC-PC- Cas9 AAGAAAUUUAAUUAGG SEQ ID NO. NTS GACUAAAACAAAGAGUU 90UGCGGGACUCUGCGGGG UUACAAUCCCCUAAAAC CGCUUUUAAAAUUCAAA UAAAUUUUGCUUUAGUUGAUAAAUUUGGUAUAU AAGGAGGGACUACAACA AAGAGUUUGCGGGACUC UGCGGGGUUACAAUCCCCUAAAACCGCUUUUAAA AUUCAAAUAAAUUUUGC UUU 8L I NASC-PC1 Cas9NNNNNNNNNNNNNNNN SEQ ID NO. 809-817 NNNNGUUUUAGAGCTAT 91GCTGTGAAAACAGCATA GCAAGUUAAAAUAAGGC UACUGCCG 8L II NASC-PC2 Cas9NNNNNNNNNNNNNNNN SEQ ID NO. 828-836 NNNNGUUUUAGAGCTAT 92GCTGTGAAAACAGCATA GCAAGUUAAAAUAAGGC UAGUCACG 8L 835-800 NASC-PC- Cas9CGGCAGUCCGUUAUCAA SEQ ID NO. NTS CUUGAAAAAGUGGCACC 93 GAGUCGGUGCUUAGUUGAUAAAUCGUGACGUCCG UUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGC UUU 9A II + INASC-PC1- Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. 2TS Cpf1 NNNNGUUUUAGAGCUAU 94GCUGUGAAAACAGCAUA GCAAGUUAAAAUAAGGC UAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUACUGAUAA UUUCUACUCUUGUAGAU NNNNNNNNNNNNNNNN NNNN 6J VIINASC-PC1- Cas9 NNNNNNNNNNNNNNNN SEQ ID NO. 2TS Cpf1 NNNNGUUUUAGAGCUAU 97GCUGUUACCAAUUGAUA GUAGAUNNNNNNNNNN NNNNNNNNNN 6J VIII NASC-PC- Cpf1AAUUUCUACUUGAUAAC SEQ ID NO. NTS Cas9 AGCAUAGCAAGUUAAAA 98UAAGGCUAUCCGUUAUC AACUUGAAAAAGUGGCA CCGAGUCGGUGCUUU 6K X NASC-PC1 Cas9NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUUUUGUACUCU 95 CAAGAUUCAAAUAACAGCAUAGCAAGUUAAAAUA AGGCUAUCCGUUAUCAA CUUGAAAAAGUGGCACC GAGUCGGUGCUUU 6KIX NASC-PC2 Cas9* NNNNNNNNNNNNNNNN SEQ ID NO. NNNNGUUUUAGAGCUAU 96GCUGUUACGUAAAUCUU GCAGAAGCUACAAAGAU AAGGCUUCAUGCCGAAA UCAACACCCUGUCAUUUUAUGGCAGGGUGUUU *S. thermophilus CRISPR-I Cas9 protein, SEQ ID NO. 108or S. thermophilus CRISPR-I dCas9 protein, SEQ ID NO. 109

Following the guidance of the present specification, one or ordinaryskill in the art can design NASC polynucleotide components (e.g., basedon other NASC polynucleotide components described herein) for differentcognate Cas proteins (e.g., C. jejuni Cas9 protein (SEQ ID NO. 103), C.jejuni dCas9 protein (SEQ ID NO. 56), S. aureus Cas9 (SEQ ID NO. 99), S.aureus dCas9 (SEQ ID NO. 102), Lachnospiraceae bacterium Cpf1 protein(SEQ ID NO. 106), Lachnospiraceae bacterium dCpf1 protein (SEQ ID NO.107) or Acidaminococcus sp. Cpf1 (SEQ ID NO. 104).

EXAMPLE 2 Production of sgRNAs and NASC Polynucleotide Components

This Example describes production of sgRNAs and NASC polynucleotidecomponents NASC-PC1 (Table 9, generic target sequence SEQ ID NO. 83) andNASC-PC2 (Table 9, generic target sequence SEQ ID NO. 84), asillustrated in FIG. 6G. The sgRNAs and NASC polynucleotide componentsdescribed in this Example were used in Cas cleavage assays (Example 5).

NASC-PC1 and NASC-PC2 comprised different first stem element nucleicacid sequences (illustrated in FIG. 6E, 608-609 and 619-620) to limitformation of secondary structures within each NASC-PC that may interferewith the formation of stable secondary structure between the NASC-PC1first stem element nucleic acid sequences and NASC-PC2 first stemelement nucleic acid sequences complementary to the first stem elementnucleic acid sequence.

Two sgRNA backbones were used (sgRNA-1 and sgRNA-2), each comprisingdifferent upper stem and lower stem nucleic acid sequences (illustratedin FIG. 2C, 221-222/227-228 and 223-224/225-226, respectively); thebulge sequences were the same.

Four nucleic acid target-binding sequences, each 20 nucleotides inlength, were selected. One of the four nucleic acid target bindingsequences was incorporated at the 5′ end of a sgRNA-1 and a sgRNA-2backbone, and the 5′ end of a NASC-PC1 and a NASC-PC2. The fourdouble-stranded DNA target sequences were as follows: Target 1 (AAVST1)corresponded to a human AAVS-1 target sequence. Target 2 (VT2), Target 3(VT3) and Target 4 (VT4) were DNA target sequences present in the vectorsequence (SEQ ID NO. 20).

RNA components were produced by in vitro transcription using a T7 QuickHigh Yield RNA Synthesis Kit (New England Biolabs, Ipswich, Mass.) froma double-stranded DNA template incorporating a T7 promoter at the 5′ endof the DNA sequences.

A double-stranded DNA template for each sgRNA, NASC-PC1, and NASC-PC2was assembled by PCR using 3′ overlapping oligonucleotide primerscontaining DNA sequences corresponding to each sgRNA, NASC-PC1, andNASC-PC2. The oligonucleotide primers are presented in Table 10.

TABLE 10 Overlapping Primers for Generation of sgRNA, NASC-PC1, andNASC-PC2 Encoding Templates Construct designation Target OligonucleotidesgRNA-1-AAVST1 target-1 SEQ ID NO. 1, 3, 11, 12, 2 sgRNA-1-VT2 target-2SEQ ID NO. 1, 4, 11, 12, 2 sgRNA-1-VT3 target-3 SEQ ID NO. 1, 5, 11, 12,2 sgRNA-1-VT4 target-4 SEQ ID NO. 1, 6, 11, 12, 2 sgRNA-2-AAVST1target-1 SEQ ID NO. 1, 7, 13, 14, 2 sgRNA-2-VT2 target-2 SEQ ID NO. 1,8, 13, 14, 2 sgRNA-2-VT3 target-3 SEQ ID NO. 1, 9, 13, 14, 2 sgRNA-2-VT4target-4 SEQ ID NO. 1, 10, 13, 14, 2 NASC-PC1-AAVST1 target-1 SEQ ID NO.1, 3, 15, 16, 2 NASC-PC1-VT2 target-2 SEQ ID NO. 1, 4, 15, 16, 2NASC-PC1-VT3 target-3 SEQ ID NO. 1, 5, 15, 16, 2 NASC-PC1-VT4 target-4SEQ ID NO. 1, 6, 15, 16, 2 NASC-PC2- AAVST1 target-1 SEQ ID NO. 1, 7,17, 18, 2 NASC-PC2-VT2 target-2 SEQ ID NO. 1, 8, 17, 18, 2 NASC-PC2-VT3target-3 SEQ ID NO. 1, 9, 17, 18, 2 NASC-PC2-VT4 target-4 SEQ ID NO. 1,10, 17, 18, 2

The DNA primers were present at a concentration of 2 nM each. One DNAprimer corresponded to the T7 promoter (SEQ ID NO. 1) and the other tothe 3′ end of the RNA sequence (SEQ ID NO. 2) and were used at aconcentration of 640 nM to drive the amplification reaction. PCRreactions were performed using Q5 Hot Start High-Fidelity 2× Master Mix(New England Biolabs, Ipswich, Mass.) following the manufacturer'sinstructions. PCR assembly reactions were carried out using thefollowing thermal cycling conditions: 98° C. for 2 minutes; 2 cycles of20 seconds at 98° C., 20 seconds at 52.5° C., 20 seconds at 72° C.;followed by 32 cycles of 20 seconds at 98° C., 20 seconds at 57° C., 20seconds at 72° C.; and a final extension at 72° C. for 2 minutes. DNAproduct quality was evaluated after the PCR reaction by agarose gelelectrophoresis (1.5%, SYBR® Safe, Life Technologies, Grand Island,N.Y.).

Between 0.25-0.5 μg of the DNA template for each sgRNA, NASC-PC1, andNASC-PC2 was used as a template for transcription using T7 High YieldRNA Synthesis Kit (New England Biolabs, Ipswich, Mass.) forapproximately 16 hours at 37° C. Transcription reactions were treatedwith DNase I (New England Biolabs, Ipswich, Mass.) and purified usingGeneJet RNA Cleanup and Concentration Kit (Life Technologies, GrandIsland, N.Y.). RNA yield was quantified using the Nanodrop™ 2000 System(Thermo Scientific, Wilmington, Del.). The quality of the transcribedRNA was checked by agarose gel electrophoresis (2%, SYBR® Safe; LifeTechnologies, Grand Island, N.Y.). The sgRNA and NASC polynucleotidecomponent sequences are shown in Table 11.

TABLE 11 sgRNA, NASC-PC1, and NASC-PC2 Sequences Name Sequence*SEQ ID NO. sgRNA- GGGGCCACUAGGGACAGGAUGUCUCAGAGCUA SEQ ID NO. 1-UGCAGUCCUGGACAACUGCCGAACAGGACUGC 25 AAVST1AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA-GUAGGCUAUAGUGUAGAUCUGUCUCAGAGCUA SEQ ID NO. 1-UGCAGUCCUGGACAACUGCCGAACAGGACUGC 26 VT2 AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA- GGAAAAAGUGGAAGCGGCGAGUCUCAGAGCUASEQ ID NO. 1- UGCAGUCCUGGACAACUGCCGAACAGGACUGC 27 VT3AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA-GGCGAUAAGUCGUGUCUUACGUCUCAGAGCUA SEQ ID NO. 1-UGCAGUCCUGGACAACUGCCGAACAGGACUGC 28 VT4 AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA- GGGGCCACUAGGGACAGGAUGCACAUGAGGAUSEQ ID NO. 2- UCUCAUGAGGGACGGCAGAAGAACCUCAUGAG 29 AAVST1AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA-GUAGGCUAUAGUGUAGAUCUGCACAUGAGGAU SEQ ID NO. 2-UCUCAUGAGGGACGGCAGAAGAACCUCAUGAG 30 VT2 AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA- GGAAAAAGUGGAAGCGGCGAGCACAUGAGGAUSEQ ID NO. 2- UCUCAUGAGGGACGGCAGAAGAACCUCAUGAG 31 VT3AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU sgRNA-GGCGAUAAGUCGUGUCUUACGCACAUGAGGAU SEQ ID NO. 2-UCUCAUGAGGGACGGCAGAAGAACCUCAUGAG 32 VT4 AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC- GGGGCCACUAGGGACAGGAUGUCUCAGAGCUASEQ ID NO. PC1- UGCAGUCCUGGACAACUGCCGAACCUCAUGAG 33 AAVST1AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC-GUAGGCUAUAGUGUAGAUCUGUCUCAGAGCUA SEQ ID NO. PC1-UGCAGUCCUGGACAACUGCCGAACCUCAUGAG 34 VT2 AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC- GGAAAAAGUGGAAGCGGCGAGUCUCAGAGCUASEQ ID NO. PC1- UGCAGUCCUGGACAACUGCCGAACCUCAUGAG 35 VT3AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC-GGCGAUAAGUCGUGUCUUACGUCUCAGAGCUA SEQ ID NO. PC1-UGCAGUCCUGGACAACUGCCGAACCUCAUGAG 36 VT4 AAUCCAAGUAUGUGUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC- GGGGCCACUAGGGACAGGAUGCACAUGAGGAUSEQ ID NO. PC2- UCUCAUGAGGGACGGCAGAAGAACAGGACUGC 37 AAVST1AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC-GUAGGCUAUAGUGUAGAUCUGCACAUGAGGAU SEQ ID NO. PC2-UCUCAUGAGGGACGGCAGAAGAACAGGACUGC 38 VT2 AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC- GGAAAAAGUGGAAGCGGCGAGCACAUGAGGAUSEQ ID NO. PC2- UCUCAUGAGGGACGGCAGAAGAACAGGACUGC 39 VT3AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUU NASC-GGCGAUAAGUCGUGUCUUACGCACAUGAGGAU SEQ ID NO. PC2-UCUCAUGAGGGACGGCAGAAGAACAGGACUGC 40 VT4 AUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU *NASC-PC hybridizing regions areunderlined

This method for production of sgRNA and NASC polynucleotide componentscan be applied to the production of other the sgRNA and NASCpolynucleotide components by one of ordinary skill in the art in view ofthe teachings of the specification.

EXAMPLE 3 Production of Double-Stranded DNA Target Sequences for Use inCleavage Assays by Cloning of Double-Stranded DNA Target Sequences intoPlasmids

Double-stranded DNA target sequences for use in the in vitro Cas proteincleavage assays were produced though the ligation of a double-strandednucleic acid target sequence (e.g., the AAVS-1 target sequence) into acloning vector backbone. Each vector was transformation into a suitablestrain of E. coli for production of double-stranded DNA targetsequences.

A 25 nucleotide single-stranded DNA target sequence corresponding to thehuman Adeno-Associated Virus Integration Site 1 (AAVS-1) was appended insilico with a randomized nucleic acid sequence of 47 nucleotides at the5′ end and a randomized nucleic acid sequence of 53 nucleotides at the3′ end. Forward and reverse oligonucleotide primers compatible with theElectra™ Vector System (DNA2.0, Newark, Calif.) were incorporated at the5′ end and the 3′ end of the DNA target sequence, producing a 237 bpsingle-stranded DNA sequence. A nucleic acid sequence of 237 bpsingle-stranded DNA (“DNA cloning fragment”), as well as nucleic acidsequences of forward and reverse amplification oligonucleotide primers,were provided to a commercial manufacturer for synthesis. Thesesingle-stranded DNA sequences are shown in Table 12.

TABLE 12 Single-stranded DNA Sequences SEQ ID Description Sequence* NO.DNA TACACGTACTTAGTCGCTGAAGCTCTTCTATG SEQ ID cloningCAAGCAGAAGACGGCATACGAGATCGAGTAA NO. 19 FragmentTGTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCTGCTACTGGGGCCACTAGGGACAGGATNGGTGCTAGCTCAGATCGGAAGAGCGTCGTGTA GGGAAAGAGTGTAGGCTATAGTGTAGATCTCGGTGGTCGCCGTATCATTGGTAGAAGAGCCGT CAATCGAGTTCGTACCT ForwardTACACGTACTTAGTCGCTGAAGCTCTTCTATG SEQ ID primer CAAGCAGAAGACGGCATACGAGATNO. 21 Reverse AGGTACGAACTCGATTGACGGCTCTTCTACCA SEQ ID primerATGATACGGCGACCACCGAGATCT NO. 22 *AAVS-1 DNA target sequence comprising aPAM is underlined

The single-stranded DNA cloning fragment was amplified via PCR togenerate double-stranded DNA for use with the Electra™ Vector System(DNA2.0, Newark, Calif.). The PCR reaction mixture was as follows: 0.5unit KAPA HiFi Hot Start DNA Polymerase (Kapa Biosystems, Wilmington,Mass.), 1× reaction buffer, 0.3 mM dNTPs, 200 nM forward primer (SEQ IDNO. 21), 200 nM reverse primer (SEQ ID NO. 22), and 80 nM of the DNAcloning fragment (SEQ ID NO. 19) in a total volume of 25 μL. The DNAcloning fragment was amplified using the following conditions: 95° C.for 4 minutes, 30 cycles of 20 seconds at 98° C., 20 seconds at 60° C.,and 30 seconds at 72° C., followed by a final extension at 72° C. for 5minutes. PCR products were purified using Spin Smart™ PCR purificationtubes (Denville Scientific, South Plainfield, N.J.) and quantified usinga Nanodrop™ 2000 UV-Vis spectrophotometer (Thermo Scientific,Wilmington, Del.).

The double-stranded DNA cloning fragment was cloned into thecommercially available “pD441-SR: T5-sRBS-ORF, Ecoli-Elec D” vector (anElectra™ bacterial DNA vector (DNA2.0, Newark, Calif.)) using themanufacturer's cloning protocol. The following cloning reaction mixturewas prepared: 20 ng of the PCR-amplified cloning fragment, 20 ng of thebacterial DNA vector, 2 μl of Electra™ buffer mix (DNA2.0, Newark,Calif.), and 1 μl of Electra™ enzyme mix (DNA2.0, Newark, Calif.) in afinal volume of 20 μL. The cloning reaction mixture was then brieflyvortexed, subjected to centrifugation using benchtop centrifuge, andincubated at room temperature for 20 minutes.

After incubation, 1 μL of One Shot® Mach1™ T1R (Thermo Scientific,Wilmington, Del.) chemically competent E. coli cells were mixed with 2μL of the cloning reaction mixture to form a transformation mixture thatwas incubated in ice for 30 minutes. The transformation mixture washeat-shocked for 30 seconds at 42° C., and incubated in ice for 2minutes. 250 μL of room temperature S.O.C. medium (Thermo Scientific,Wilmington, Del.) was added to the transformation mixture, and themixture was incubated at 37° C. for 1 hour with shaking. After thisincubation, 50 μL of the cell mixture was spread onto an LB agar platewith 50 μg/mL kanamycin, and the plate was incubated overnight at 37° C.for bacterial colony formation.

Five bacterial colonies were picked and transferred to separate 15 mLculture tubes containing 5 mL of LB supplemented with 50 μg/mL kanamycinculture medium and the tubes were incubated for 8 hours with shaking.Cells were pelleted by centrifugation at 4000 RPM for 15 minutes,culture medium was aspirated, and the cells were re-suspended in 200 μLof LB culture medium without antibiotics. DNA vectors were extractedfrom the bacteria of each of the five bacterial colonies using QlAprepSpin Miniprep Kit (Qiagen, Venlo, Netherlands) following themanufacturer's instructions. DNA vector yields were quantified using aNanodrop™ 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington,Del.). 250 ng of each DNA vector was Sanger sequenced to verifyincorporation of the DNA cloning fragment corresponding to SEQ ID NO.19. The full DNA vector sequence, including AAVS-1 target sequence, isprovided as SEQ ID NO. 20.

A bacterial clone identified as containing a DNA vector comprising theDNA cloning fragment was cultured in 100 mL of LB supplemented with 50μg/mL kanamycin culture medium and grown overnight at 37° C. withshaking. Cells were pelleted by centrifugation at 4000 RPM for 15minutes, culture medium was aspirated, and the DNA vector was purifiedusing a QIAprep Spin Maxiprep Kit (Qiagen, Venlo, Netherlands) followingthe manufacturer's instructions. DNA vector yields were quantified usinga Nanodrop™ 2000 UV-Vis spectrophotometer (Thermo Scientific,Wilmington, Del.).

The DNA vector was prepared to be used in Cas cleavage assay bylinearization of the circular vector with an Ascl Type II restrictionendonuclease. To linearize the circular DNA vector, the followingreaction mixture was assembled: 1 unit of Ascl restriction endonuclease(New England Biolabs, Ipswich, Mass.) per 1 μg of circular DNA vector,and 1× CutSmart® buffer (New England Biolabs, Ipswich, Mass.) in a finalvolume of 50 uL. The reaction mixture was incubated for 1 hour at 37°C., and the reaction stopped by incubation at 80° C. for 20 minutes.Linear DNA vector was purified using the QIAquick PCR Purification Kit(Qiagen, Venlo, Netherland) following the manufacturer's instructions.Linear DNA vector yields were quantified using a Nanodrop™ 2000 UV-Visspectrophotometer (Thermo Scientific, Wilmington, Del.).

Other suitable cloning methods and DNA vectors can be used for theincorporation of double-stranded DNA target sequences followingessentially the method described in this Example. If linearization ofthe DNA vector is undesirable or unnecessary, a circular DNA vector canbe used in Cas cleavage assays.

EXAMPLE 4 Production of Double-Stranded DNA Target Sequences for Use inCleavage Assays Using PCR

Double-stranded DNA target sequences for use in in vitro Cas proteincleavage assays can be produced using PCR amplification of selectednucleic acid target sequences from genomic human DNA.

Genomic human DNA comprising the Adeno-Associated Virus Integration Site1 (AAVS-1) can be prepared by phenol-chloroform extraction from humancell line K562 (American Type Culture Collection (ATCC), Manassas, Va.).PCR reactions can be carried out with Q5 Hot Start High-Fidelity 2×Master Mix (New England Biolabs, Ipswich, Mass.) following themanufacturer's instructions. 20 ng/μL gDNA in a final volume of 25 μlcan be used to amplify the selected nucleic acid target sequence underthe following conditions: 98° C. for 2 minutes, 35 cycles of 20 secondsat 98° C., 20 seconds at 60° C., 20 seconds at 72° C., and a finalextension at 72° C. for 2 minutes. PCR products can be purified usingSpin Smart™ PCR purification tubes (Denville Scientific, SouthPlainfield, N.J.) and can be quantified using a Nanodrop™ 2000 UV-Visspectrophotometer (Thermo Scientific, Wilmington, Del.).

Examples of forward and reverse primers that can be used foramplification of the AAVS-1 DNA target sequences from gDNA are presentedin Table 13.

TABLE 13 AAVS-1 DNA Target Sequence Oligonucleotide Primers SEQ ID NO.Sequence SEQ ID NO. 23 CCCCGTTCTCCTGTGGATTC SEQ ID NO. 24ATCCTCTCTGGCTCCATCGT

The AAVS-1 DNA target sequence can be amplified using SEQ ID NO. 23 andSEQ ID NO. 24 to produce a 495 bp double-stranded AAVS-1 DNA targetsequence.

Other suitable double-stranded DNA target sequences can be obtainedusing essentially the same method by choosing suitable oligonucleotideprimers. gDNA from the any organism (e.g., plant, bacteria, yeast,algae, and the like) can be used instead of DNA derived from humancells. Furthermore, DNA target sequences can be amplified via PCR frompolynucleotides other than gDNA (e.g., vectors and gel isolated DNAfragments).

EXAMPLE 5 Cas Cleavage Assays

This Example illustrates the use of a NASC polynucleotide compositionsand a Cas9 protein in an in vitro assay to evaluate cleavage percentagesof nucleic acid target sequences by the NASC polynucleotidecompositions.

NASC-PC1 and NASC-PC2 comprised different first stem element nucleicacid sequences to limit formation of secondary structures within eachNASC-PC that may interfere with the formation of stable secondarystructure through hydrogen bond formation between the NASC-PC1 firststem element nucleic acid sequences and NASC-PC2 first stem elementnucleic acid sequences complementary to the NASC-PC1 first stem elementnucleic acid sequence.

The generic components of the NASC polynucleotide composition used inthis Example were NASC-PC1 (Table 9, generic target sequence SEQ ID NO.83) and NASC-PC2 (Table 9, generic target sequence SEQ ID NO. 84). Thegeneral structure of this NASC-P1/NASC-P2 pair is illustrated in FIG.6G.

Ribonucleoprotein complexes of sgRNA/Cas9 protein andNASC-PC1/NASC-PC2/Cas9 protein were used in in vitro Cas9 cleavageassays to evaluate percent cleavage of each complex relative to thecorresponding double-stranded DNA target sequences on a DNA vector.

The ribonucleoprotein complexes of sgRNA/Cas9 protein andNASC-PC1/NASC-PC2/Cas9 used the sgRNA and NASC-PC1/NASC-P2 constructsset forth in Example 2 Table 11. Target 1 (AAVST1) corresponded to ahuman AAVS-1 target sequence. Target 2 (VT2), Target 3 (VT3) and Target4 (VT4) were DNA target sequences present in the vector sequence (SEQ IDNO. 20). In a cleavage reaction with only a single sgRNA or single NASCpolynucleotide component, the linearized vector was used. In a cleavagereaction with two sgRNAs or NASC-PC1/NASC-PC2 components, the circularvector was used. Cleavage of the linear plasmid with a sgRNA yielded twoDNA fragments, cleavage of the circular plasmid with two sgRNA orNASC-PC1/NASC-PC2 components yielded two DNA target fragments. The sizeof the double-stranded DNA target sequences and the sizes of thepredicted cleavage fragments are presented in Table 14.

TABLE 14 Target and Cleavage Fragment Sizes DNA target Fragment 1Fragment 2 Target vector (bp) (bp) AAVST1 Linear 1706 2469 VT2 Linear1769 2406 VT3 Linear 3214 961 VT4 Linear 350 3825 AAVST1/VT3 Circular1509 2666 AAVST1/VT4 Circular 1357 2818 VT2/VT3 Circular 1446 2729VT2/VT4 Circular 1420 2755

The sgRNA and NASC-PC1/NASC-PC2 components were diluted to a suitableworking concentration. sgRNA and NASC-PC components were aliquoted intoseparate tubes to a final concentration of 50 nM. Pairs of sgRNAs andNASC-PC1/NASC-PC2 components were aliquoted into separate tubes to afinal concentration of 50 nM for each component. All RNAs were incubatedfor 2 minutes at 95° C., removed from thermocycler, and allowed toequilibrate to room temperature. The combinations of the sgRNA andNASC-PC1/NASC-PC2 components that were used in cleavage reactions arepresented in Table 15.

TABLE 15 sgRNA and NASC-PC1/NASC-PC2 Reaction Mixture Components SEQ IDSEQ ID Reaction RNA-1 type NO. RNA-2 type NO. 1 sgRNA-1-AAVST1 SEQ ID —— NO. 25 2 sgRNA-1-VT2 SEQ ID — — NO. 26 3 sgRNA-1-VT3 SEQ ID — — NO. 274 sgRNA-1-VT4 SEQ ID — — NO. 28 5 sgRNA-2-AAVST1 SEQ ID — — NO. 29 6sgRNA-2-VT2 SEQ ID — — NO. 30 7 sgRNA-2-VT3 SEQ ID — — NO. 31 8sgRNA-2-VT4 SEQ ID — — NO. 32 9 NASC-PC1-AAVST1 SEQ ID — — NO. 33 10NASC-PC1-VT2 SEQ ID — — NO. 34 11 NASC-PC1-VT3 SEQ ID — — NO. 35 12NASC-PC1-VT4 SEQ ID — — NO. 36 13 NASC-PC2- AAVST1 SEQ ID — — NO. 37 14NASC-PC2-VT2 SEQ ID — — NO. 38 15 NASC-PC2-VT3 SEQ ID — — NO. 39 16NASC-PC2-VST4 SEQ ID — — NO. 40 17 sgRNA-1-AAVST1 SEQ ID sgRNA-2-VT3 SEQID NO. 25 NO. 31 18 NASC-PC1-AAVST1 SEQ ID NASC-PC2-VT3 SEQ ID NO. 33NO. 39 19 sgRNA-1-AAVST1 SEQ ID sgRNA-2-VT4 SEQ ID NO. 25 NO. 32 20NASC-PC1-AAVST1 SEQ ID NASC-PC2-VT4 SEQ ID NO. 33 NO. 40 21 sgRNA-1-VT2SEQ ID sgRNA-2-VT3 SEQ ID NO. 26 NO. 31 22 NASC-PC1-VT2 SEQ IDNASC-PC2-VT3 SEQ ID NO. 34 NO. 39 23 sgRNA-1-VT2 SEQ ID sgRNA-2-VT4 SEQID NO. 26 NO. 32 24 NASC-PC1-VT2 SEQ ID NASC-PC2-VT4 SEQ ID NO. 34 NO.40

Each sgRNA reaction mixture component(s) and NASC-PC1/NASC-PC2 reactionmixture component(s) was added to a Cas9 reaction mix. S. pyogenes Cas9protein was recombinantly expressed in E. coli and purified for use inthe in vitro biochemical cleavage assay. The Cas9 reaction mixturecomprised Cas9 protein diluted to a final concentration of 200 nM inreaction buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, and 5% glycerol atpH 7.4). Each Cas9 reaction mixture was incubated at 37° C. for 10minutes. Cleavage in each Cas9 reaction mixture was initiated byaddition of the DNA target vector to a final concentration of 5 nM. EachCas9 reaction mixture was mixed, centrifuged briefly, and incubated for15 minutes at 37° C. The cleavage reaction was terminated by theaddition of Proteinase K (Denville Scientific, South Plainfield, N.J.)at a final concentration of 0.2 μg/μL and 0.44 mg/μL RNase A Solution(Sigma Aldrich, St. Louis, Mo.) to each Cas9 reaction mixture.

Each Cas9 reaction mixture was then incubated for 25 minutes at 37° C.and 25 minutes at 55° C. Each Cas9 reaction mixture was evaluated forcleavage activity using the Fragment Analyzer™ (Advanced AnalyticalTechnologies, Ames, Iowa) System and the DNF-474-05000 High SensitivityNGS Reagent Kit (Advanced Analytical Technologies, Ames, Iowa). The datafrom the Fragment Analyzer™ System provided the concentration of eachcleavage fragment and of the DNA target vector that remained aftercleavage for each Cas9 reaction mixture. For each Cas9 reaction mixture,percent cleavage was calculated by dividing the sum of the cleavagefragments by the sum of both the cleavage fragments and the DNA targetvector that remained after cleavage.

Table 16 presents the cleavage data for each of the ribonucleoproteincomplexes of sgRNA/Cas9 protein and NASC-PC1/NASC-PC2/Cas9.

TABLE 16 Biochemical Cleavage of DNA Target Sequences with sgRNA/Cas9Protein Complexes and NASC- PC1/NASC-PC2/Cas9 Protein Complexes Per-cent Reac- SEQ ID SEQ ID cleav- tion NO. RNA-1 type NO. RNA-2 type age 1SEQ ID sgRNA-1- — — 100%  NO. 25 AAVST1 2 SEQ ID sgRNA-1-VT2 — — 95% NO.29 3 SEQ ID sgRNA-1-VT3 — — 92% NO. 26 4 SEQ ID sgRNA-1-VT4 — — 94% NO.30 5 SEQ ID sgRNA-2- — — 100%  NO. 27 AAVST1 6 SEQ ID sgRNA-2-VT2 — —100%  NO. 31 7 SEQ ID sgRNA-2-VT3 — — 96% NO. 28 8 SEQ ID sgRNA-2-VT4 —— 95% NO. 32 9 SEQ ID NASC-PC1- — —  LOD* NO. 33 AAVST1 10 SEQ IDNASC-PC1-VT2 — — LOD NO. 37 11 SEQ ID NASC-PC1-VT3 — — LOD NO. 34 12 SEQID NASC-PC1-VT4 — — LOD NO. 38 13 SEQ ID NASC-PC2- — — LOD NO. 35 AAVST114 SEQ ID NASC-PC2-VT2 — — LOD NO. 39 15 SEQ ID NASC-PC2-VT3 — — LOD NO.36 16 SEQ ID NASC-PC2-VT4 — — LOD NO. 40 17 SEQ ID sgRNA-1- SEQ IDsgRNA-2-VT3 88% NO. 25 AAVST1 NO. 31 18 SEQ ID NASC-PC1- SEQ IDNASC-PC2-VT3 89% NO. 33 AAVST1 NO. 39 19 SEQ ID sgRNA-1- SEQ IDsgRNA-2-VT4 91% NO. 25 AAVST1 NO. 32 20 SEQ ID NASC-PC1- SEQ IDNASC-PC2-VT4 92% NO. 33 AAVST1 NO. 40 21 SEQ ID sgRNA-1-VT2 SEQ IDsgRNA-2-VT3 81% NO. 26 NO. 31 22 SEQ ID NASC-PC1-VT2 SEQ ID NASC-PC2-VT386% NO. 34 NO. 39 23 SEQ ID sgRNA-1-VT2 SEQ ID sgRNA-2-VT4 94% NO. 26NO. 32 24 SEQ ID NASC-PC1-VT2 SEQ ID NASC-PC2-VT4 96% NO. 34 NO. 40 *LODindicates cleavage values below the limit of detection

The data presented in Table 16 demonstrates that eachNASC-PC1/NASC-PC2/Cas9 protein complex of Reactions 18, 20, 22, and 24(Table 16) facilitated Cas protein mediated site-specific cleavage ofthe two DNA target sequences corresponding to the two nucleic acidtarget binding sequences of the NASC-PC1/NASC-PC2/Cas9 protein complex.Furthermore, the percent of site-specific cleavage by eachNASC-PC1/NASC-PC2/Cas9 protein complex was essentially equivalent tosite-specific cleavage of the same two DNA target sequences by twosgRNA/Cas9 protein complexes, wherein one sgRNA/Cas9 protein complextargeted cleavage at a first DNA target sequence and a second sgRNA/Cas9protein complex targeted cleavage at a second DNA target sequence(compare percent cleavage of reactions 17 with 18; 19 with 20; 21 with22; and 23 with 24). The data presented in Table 16 also demonstrateseach NASC-PC1 was required to be paired with a complementary NASC-PC2 inorder to target site-specific cleavage by the associated Cas9 proteins(see Table 16, Reactions 9-16); that is, an individual polynucleotidecomponent of a NASC polynucleotide composition was incapable ofsupporting Cas protein mediated site-specific cleavage.

Following the guidance of the present specification and Examples, thebiochemical cleavage assay described in this Example can be practiced byone of ordinary skill in the art with other NASC polynucleotidecompositions and cognate Cas proteins (e.g., Cas9 proteins and Cpf1proteins).

EXAMPLE 6 Deep Sequencing Analysis for Detection of Target SequenceModifications in Eukaryotic Cells

This Example illustrates the use of deep sequencing analysis to evaluateand compare the percent cleavage in cells using NASC polynucleotidecomposition/Cas protein complexes relative to selected double-strandedDNA target sequences.

A. Genome Target Sequence Selection

Two target nucleic acid sequences can be selected from exonic regions inthe human genome (e.g., X-Ray Repair Cross Complementing 5 (XRCC5) genesequence). Nucleic acid sequences twenty nucleotides in length that are5′ adjacent to PAM sequences (e.g., a S. pyogenes Cas9 PAM 5′—NGG) canbe selected, for example, the XRCC5 target DNA sequences presented inTable 17.

TABLE 17 XRCC5 Target DNA Sequences hg38 Target chromosomal SEQ ID nameTarget sequence coordinate NO. XRCC5T1 GGTGGACAAGCGGCAGATAG chr2: SEQ ID216109346- NO. 41 216109365 XRCC5T3 GCACCATGTTGCCGGTCCTC chr2: SEQ ID216109421- NO. 42 216109440

B. Construction of NASC Polynucleotide Compositions

A NASC polynucleotide composition comprising NASC-PC1 and NASC-PC2 canbe used. Nucleic acid target binding sequences corresponding to XRCC5T1can be incorporated at the 5′ end of NASC-PC1, and nucleic acid targetbinding sequences corresponding to XRCC5T3 can be incorporated at the 5′end of NASC-PC2. As positive controls, a nucleic acid target bindingsequence corresponding to XRCC5T1 can be incorporated at the 5′ end of asgRNA, and a nucleic acid target binding sequences corresponding toXRCC5T3 can be incorporated at the 5′ end of a sgRNA. NASC-PC1,NASC-PC2, and sgRNAs can be produced as described in Example 2. Examplesof sequences for NASC-PC1, NASC-PC2, and sgRNAs are given in Table 18.

TABLE 18 sgRNA and NASC Polynucleotide Component Sequences Component RNASEQ ID designation Type RNA sequence* NO. sgRNA- sgRNAGGUGGACAAGCGGCAGAUAGGUUU SEQ ID XRCC5-T1 UAGAGCUAUGCUGUUUUGGAAACA NO. 43AAACAGCAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCUUsgRNA- sgRNA GCACCAUGUUGCCGGUCCUCGUUUU SEQ ID XRCC5-T3AGAGCUAUGCUGUUUUGGAAACAA NO. 44 AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUU NASC-PC1- NASC₁GGUGGACAAGCGGCAGAUAGGUUU SEQ ID XRCC5-T1 UAGAGCUAUGCUGUUUUGGAAACU NO. 45UUUCAGCACGAUAAGUUAUUAUAA GGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCUUNASC-PC2- NASC_(1C) GCACCAUGUUGCCGGUCCUCGUAAU SEQ ID XRCC5-T3AGAAUCGUGCUGAAAAGGAAACAA NO. 46 AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUU *NASC-PC hybridizingregions are underlined

C. Formation of NASC/Cas9 Protein Nucleoprotein Complexes

S. pyogenes Cas9 can be C-terminally tagged with two nuclearlocalization sequences (NLS) and can be recombinantly expressed in E.coli, and purified using chromatographic methods. Ribonucleoproteincomplexes can be formed at a concentration of 80 pmol Cas9 protein:120pmol NASC-PC1:120 pmols NASC-PC2. Control sgRNA components can beindividually assembled into ribonucleoprotein complexes with Cas9protein in a similar manner. Prior to assembly with the Cas9 protein,NASC-PC1, NASC-PC2, and the sgRNAs can be diluted to the desiredconcentration (120 pmol) in a final volume of 2 μL, incubated for 2minutes at 95° C., removed from the thermocycler, and allowed toequilibrate to room temperature. The Cas9 protein can be diluted to anappropriate concentration in binding buffer (20 mM HEPES, 100 mM KCl, 5mM MgCl₂, and 5% glycerol at pH 7.4) to a final volume of 3 μL and canbe mixed with the 2 μL of each NASC-PC1, NASC-PC2, and the sgRNAsfollowed by incubation at 37° C. for 30 minutes.

D. Cell Transfections Using the NASC/Cas9 Ribonucleoprotein Complexes

Ribonucleoprotein complexes can be transfected into HEK293 cells (ATCC,Manassas Va.), using the Nucleofector® 96-well Shuttle System (Lonza,Allendale, N.J.) following the manufacturer's protocol.Ribonucleoprotein complexes can be dispensed in a 5 μL final volume intoindividual wells of a 96-well plate, wherein the wells contain theHEK293 cells in culture medium. The cell culture medium can be removedfrom the wells of the plate and the cells can be detached with TrypLE™enzyme (Thermo Scientific, Wilmington, Del.). Suspended HEK293 cells canbe pelleted by centrifugation for 3 minutes at 200×g, TrypLE reagentscan be aspirated, and cells can be washed with calcium andmagnesium-free phosphate buffered saline (PBS). Cells can be pelleted bycentrifugation for 3 minutes at 200×g the PBS aspirated and the cellpellet can be re-suspended in 10 mL of calcium and magnesium-free PBS.

The cells can be counted using the Countess® II Automated Cell Counter(Life Technologies, Grand Island, N.Y.). 2.2×10⁷ cells can betransferred to a 1.5 ml microfuge tube and pelleted. The PBS can beaspirated and the cells can be re-suspended in Nucleofector™ SF solution(Lonza, Allendale, N.J.) to a density of 1×10⁷ cells/mL. 20 μL of thecell suspension can be added to each individual well containing 5 μL ofribonucleoprotein complexes, and the entire volume from each well can betransferred to a well of a 96-well Nucleocuvette™ Plate (Lonza,Allendale, N.J.). The plate can be loaded onto the Nucleofector™ 96-wellShuttle™ (Lonza, Allendale, N.J.) and cells can be nucleofected usingthe 96-CM-130 Nucleofector™ program (Lonza, Allendale, N.J.).Post-nucleofection, 70 μL Dulbecco's Modified Eagle Medium (DMEM; ThermoScientific, Wilmington, Del.) supplemented with 10% Fetal Bovine Serum(FBS; Thermo Scientific, Wilmington, Del.), penicillin, and streptomycin(Life Technologies, Grand Island, N.Y.) can be added to each well andthen 50 μL of the cell suspension can be transferred to a 96-well cellculture plate containing 150 μL pre-warmed DMEM complete culture medium.The plate can be then transferred to a tissue culture incubator andmaintained at 37° C. in 5% CO₂ for 48 hours.

E. Double-Stranded DNA Target Sequence Generation for Deep Sequencing

gDNA can be isolated from the HEK293 cells 48 hours after transfectionof the ribonucleoprotein complexes using 50 μL QuickExtract DNAExtraction solution (Epicentre, Madison, Wis.) per well, followed byincubation at 37° C. for 10 minutes, 65° C. for 6 minutes and 95° C. for3 minutes to stop the reaction. The isolated gDNA can be diluted with 50μL sterile water and samples can be stored at −80° C.

Using the isolated gDNA, a first PCR can be performed using Q5 Hot StartHigh-Fidelity 2× Master Mix (New England Biolabs, Ipswich, Mass.) at 1×concentration, primers at 0.5 μM each, 3.75 μL of gDNA in a final volumeof 10 μL and amplified 98° C. for 1 minute, 35 cycles of 10 seconds at98° C., 20 seconds at 60° C., 30 seconds at 72° C., and a finalextension at 72° C. for 2 minutes. Primers can be designed to amplifyeither the XRCC5_T1 region (e.g., SEQ ID NO. 47 and SEQ ID NO. 48) orXRCC5_T3 (SEQ ID NO. 49 and SEQ ID NO. 50). gDNA prepped from theNASC-PC1/NASC-PC2/Cas9 nucleofected samples and the sgRNA/Ca9nucleofected samples can be amplified with both primer pairs,separately, to assess editing of each target site by theribonucleoproteins. Each PCR reaction can be diluted 1:100 in water.

A “barcoding” PCR can be set up using unique index primers for eachsample to facilitate multiplex sequencing. Examples of such primer pairsare shown in Table 19.

TABLE 19 Barcoding Primers ID Sample Primer BARCODING PRIMERXRCC5_T1-sgRNA SEQ ID NO. 51, 52 set-1 BARCODING PRIMER XRCC5_T3-sgRNASEQ ID NO. 51, 53 set-2 BARCODING PRIMER XRCC5-T1 NASC-PC1 SEQ ID NO.51, 54 set-3 BARCODING PRIMER XRCC5-T13 NASC-PC1 SEQ ID NO. 51, 55 set-4

The barcoding PCR can be performed using Q5 Hot Start High-Fidelity 2×Master Mix (New England Biolabs, Ipswich, Mass.) at 1× concentration,primers at 0.5 μM each (Table 19), 1 μL of 1:100 diluted first PCR, in afinal volume of 10 μL, and can be amplified 98° C. for 1 minutes, 12cycles of 10 seconds at 98° C., 20 seconds at 60° C., 30 seconds at 72°C., and a final extension at 72° C. for 2 minutes.

F. SPRIselect Clean-Up

All the barcoding PCR reactions can be pooled and transferred into asingle microfuge (“amplicon library”) tube for SPRIselect bead-basedcleanup (Beckman Coulter, Pasadena, Calif.) of amplicons for sequencing.

To each tube, 0.9× volumes of SPRIselect beads can be added, mixed, andincubated at room temperature for 10 minutes. The microfuge tube can beplaced on magnetic tube stand (Beckman Coulter, Pasadena, Calif.) untilthe solution clears. Supernatant can be removed and discarded, and theresidual beads can be washed with 1 volume of 85% ethanol, and incubatedat room temperature for 30 seconds. After incubation, ethanol can beaspirated and beads can be air dried at room temperature for 10 minutes.Each microfuge tube can be removed from the magnetic stand and 0.25×volumes of Qiagen EB buffer (Qiagen, Venlo, Netherlands) can be added tothe beads, mixed vigorously, and incubated for 2 minutes at roomtemperature. Each microfuge tube can be returned to the magnet,incubated until the solution had cleared, and then the supernatantcontaining the purified amplicons can be dispensed into a cleanmicrofuge tube. The purified amplicon library can be quantified usingthe Nanodrop™ 2000 System (Thermo Scientific, Wilmington, Del.) andlibrary quality can be analyzed using the Fragment Analyzer™ System(Advanced Analytical Technologies, Ames, Iowa) and the DNF-910Double-stranded DNA Reagent Kit (Advanced Analytical Technologies, Ames,Iowa).

G. Deep Sequencing Set-Up

The pooled amplicon library can be normalized to a 4 nM concentration ascalculated from quantified values and the average size of the amplicons.The amplicon library can be analyzed on MiSeq Sequencer (Illumina, SanDiego, Calif.) with MiSeq Reagent Kit v2 (Illumina, San Diego, Calif.)for 300 cycles with two 151-cycle paired-end runs plus two eight-cycleindex reads.

H. Deep Sequencing Data Analysis

The identities of products in the sequencing data can be determinedbased on the index barcode sequences adapted onto the amplicons in thebarcoding PCR. A computational script can be used to process the MiSeqdata that executes, for example, the following tasks:

-   -   Reads can be aligned to the human genome (build GRCh38/38) using        Bowtie (bowtie-bio.sourceforge.net/index.shtml) software.    -   Aligned reads can be compared to the expected wild-type locus        region (e.g., XRCC5_T1 or XRCC5_T3)    -   Locus sequence and reads not aligning to any part of the target        locus can be discarded.    -   Reads matching wild-type target locus sequences can be tallied.    -   Reads with indels (insertion or deletion of bases) can be        categorized by indel type and tallied.    -   Total indel reads can be divided by the sum of wild-type reads        and indel reads to give percent-mutated reads.

Through the identification of indel sequences at the regions targeted bythe NASC-PC1NASC-PC2/Cas9 protein ribonucleoprotein complexes and thesgRNA/Cas9 protein ribonucleoprotein complexes, sequence-specifictargeting of in a human cell line can be determined. Editing inNASC-PC1/NASC-PC2 samples can be compared to the editing efficiencies ofsgRNA controls.

Following the guidance of the present specification and Examples, the incell editing of a genomic sequence can be practiced by one of ordinaryskill in the art with other Cas proteins and their cognate the NASCpolynucleotide compositions.

EXAMPLE 7 Identification and Screening of crRNAs

In this Example, a method is described through which crRNAs of specieshaving a Class 2 CRISPR system can be identified. The method presentedhere is adapted from Chylinski, K., et al., RNA Biology 10(5):726-737(2013). Not all of the following steps are required for screening normust the order of the steps be as presented.

A. Identify a Species Containing a Class 2 CRISPR Locus

Using the Basic Local Alignment Search Tool (BLAST,blast.ncbi.nlm.nih.gov/Blast.cgi), a search of the genomes of variousspecies can be conducted to identify Class 2 CRISPR Cas nucleases,(e.g., Cas9 protein, Cpf1 protein, Cas9-like proteins, Cpf1-likeproteins, etc.). Class 2 CRISPR systems exhibit a high diversity insequence across species, however Class 2 CRISPR nuclease orthologs haveconserved domains, for example, an HNH endonuclease domain and/or aRuvC/RNase H domain. Primary BLAST results can be filtered foridentified domains, incomplete or truncated sequences can be discarded,and species having Class 2 CRISPR nuclease orthologs can be identified.

If a Class 2 CRISPR nuclease ortholog can be identified in a species,sequences adjacent to the Cas protein ortholog coding sequence (e.g., aCas9 protein or a Cpf1 protein) can be probed for other Cas proteins andan associated repeat-spacer array to identify all sequences belonging tothe CRISPR-Cas locus. This may be done by alignment to other known Class2 CRISPR loci.

Once the sequence of the Class 2 CRISPR locus for the nuclease orthologcan be identified for the species, in silico predictive screening can beused to extract the crRNA sequence. The crRNA sequence is containedwithin CRISPR repeat array and can be identified by its hallmarkrepeating sequences interspaced by foreign spacer sequences.

B. Preparation of RNA-Seq Library

The putative CRISPR array containing the individual crRNA identified insilico can be further validated using RNA sequencing (RNA-seq).

Cells from species identified as comprising putative crRNA can beprocured from a commercial repository (e.g., ATCC, Manassas, Va.; GermanCollection of Microorganisms and Cell Cultures GmbH (DSMZ),Braunschweig, Germany).

Cells can be grown to mid-log phase and total RNA prepped using Trizolreagent (Sigma Aldrich, St. Louis, Mo.) and treated with DNasel(Fermentas, Vilnius, Lithuania).

10 μg of the total RNA can be treated with Ribo-Zero rRNA Removal Kit(Illumina, San Diego, Calif.) and the remaining RNA purified using RNAClean and Concentrators (Zymo Research, Irvine, Calif.).

A library can be then prepared using a TruSeq Small RNA LibraryPreparation Kit (Illumina, San Diego, Calif.) following themanufacturer's instructions. This results in cDNAs having adaptersequences.

The resulting cDNA library can be sequenced using MiSeq Sequencer(Illumina, San Diego, Calif.).

C. Processing of Sequencing Data

Sequencing reads of the cDNA library can be processed, for example,using the following method.

Adapter sequences can be removed using cutadapt 1.1(pypi.python.org/pypi/cutadapt/1.1) and about 15 nt can be trimmed fromthe 3′ end of the read to improve read quality.

Reads can be aligned to the genome of the respective species (i.e., fromwhich the putative crRNA was identified) using Bowtie 2(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).

The Sequence Alignment/Map (SAM) file, generated by Bowtie 2, can beconverted into a Binary Alignment/Map (BAM) file using SAMTools(samtools.sourceforge.net/) for subsequent sequencing analysis steps.

Read coverage mapping to the CRISPR locus or loci, can be calculatedfrom the BAM file using BedTools (bedtools.readthedocs.org/en/latest/).

The BED file, generated in the previous step, can be loaded intoIntegrative Genomics Viewer (IGV; www.broadinstitute.org/igv/) tovisualize the sequencing read pileup. Read pile-ups can be used toidentify the 5′ and 3′ ends of the transcribed putative crRNA sequence.

The RNA-seq data can be used to validate that a putative crRNA elementsequence is actively transcribed in vivo. Confirmed hits from comparisonof the in silico and RNA-seq screens can be validated for functionalability to support Class 2 CRISPR nuclease cleavage of a double-strandedDNA target nucleic acid sequences using the methods outline herein(e.g., Examples 2, 3, and 5). It is known in the art that Class 2 Type VCRISPR systems only requires a crRNA to facilitate Cpf1 nucleasecleavage of a double-stranded DNA target sequence, whereas Class 2 TypeII CRISPR systems require a crRNA and a cognate tracrRNA to facilitateCas9 nuclease cleavage of a double-stranded DNA target sequence.

Following the guidance of the present specification and Examples, theidentification of crRNA sequences associated with Cas9 proteins can bepracticed by one of ordinary skill in the art.

EXAMPLE 8 Identification and Screening of tracrRNAs

This Example illustrates a method by which tracrRNAs of species having,for example, a Class 2 Type II CRISPR-Cas9 system can be identified.This is adapted from Chylinski, K., et al., RNA Biology 10(5):726-737(2013). Not all of the following steps are required for screening normust the order of the steps be as presented.

A. Identify a Species Containing a CRISPR-Cas9 Type-II System

Using the Basic Local Alignment Search Tool (BLAST,blast.ncbi.nlm.nih.gov/Blast.cgi), a search of the genomes of variousspecies can be conducted to identify a Cas9 protein. Class 2 Type IICRISPR-Cas9 systems exhibit a high diversity in sequence across species,however Cas9 orthologs exhibit conserved domain architectures of acentral HNH endonuclease domain and a split RuvC/RNase domain. PrimaryBLAST results can be filtered for identified domains; incomplete ortruncated sequences can be discarded and Cas9 orthologs can beidentified.

If a Cas9 ortholog can be identified in a species, sequences adjacent tothe Cas9 ortholog-coding sequence can be probed for other Cas proteinsand a Cas-associated repeat-spacer array to identify all sequencesbelonging to the CRISPR-Cas9 locus. This may be done by alignment toother known Class 2 Type II CRISPR-Cas9 loci, with the knowledge thatclosely related species exhibit similar CRISPR-Cas9 locus architecture(e.g., Cas protein composition, size, orientation, location of array,location of tracrRNA, etc.). The tracrRNA element is typically containedwithin the Class 2 Type II CRISPR-Cas9 locus and can be readilyidentified by its sequence complementarity to the repeat elements in therepeat-spacer array. The tracr sequences complementary to the repeatelements are called the tracr anti-repeat sequences.

Once the sequence of the CRISPR-Cas9 locus corresponding to the Cas9ortholog is identified for a species, in silico predictive screening canbe used to extract the tracr anti-repeat sequence to identify theCas-associated tracrRNA. Putative anti-repeats can be screened, forexample, as follows.

If the repeat sequence is from a known species, the repeat sequence canbe identified in and retrieved from the CRISPRdb database(crispr.u-psud.fr/crispr/). If the repeat sequence is not from a knownspecies, the repeat sequence can be predicted employing CRISPRfindersoftware (crispr.u-psud.fr/Server/) using the Class 2 Type IICRISPR-Cas9 locus for the species as described above.

The identified repeat sequence for the species can be used to probe theCRISPR-Cas9 locus for the anti-repeat sequence (e.g., using the BLASTpalgorithm or the like). The search is typically restricted to intergenicregions of the CRISPR-Cas9 locus.

An identified tracr anti-repeat region can be validated forcomplementarity to the identified repeat sequence.

A putative anti-repeat region can be probed in the regions 5′ and 3′ ofthe putative anti-repeat region for the presence of a Rho-independenttranscriptional terminator (TransTerm HP, transterm.cbcb.umd.edu/).

By combining the identified sequence comprising the anti-repeat elementand the Rho-independent transcriptional terminator the sequence can bedetermined to be the putative tracrRNA of the given species.

B. Preparation of RNA-Seq Library

The in silico identified, putative tracrRNA can be further validatedusing RNA sequencing (RNA-seq).

Cells from species comprising the putative tracrRNA can be procured froma commercial repository (e.g., ATCC, Manassas Va.; DSMZ, Braunschweig,Germany).

Cells can be grown to mid-log phase and total RNA prepared using Trizolreagent (Sigma Aldrich, St. Louis, Mo.) and treated with DNaseI(Fermentas, Vilnius, Lithuania).

10 ug of the total RNA can be treated using a Ribo-Zero rRNA Removal Kit(Illumina, San Diego, Calif.) and the remaining RNA purified using RNAClean and Concentrators (Zymo Research, Irvine, Calif.).

A library can be prepared using a TruSeq Small RNA Library PreparationKit (Illumina, San Diego, Calif.) following the manufacturer'sinstructions. This results in cDNAs having adapter sequences.

The resulting cDNA library can be sequenced using a MiSeq Sequencer(Illumina, San Diego, Calif.).

C. Processing of Sequencing Data

Sequencing reads of the cDNA library can be processed, for example,using the following method.

Adapter sequences can be removed using cutadapt 1.1(pypi.python.org/pypi/cutadapt/1.1) and about 15 nt can be trimmed fromthe 3′ end of the read to improve read quality.

Reads can be aligned to the genome of the respective species (i.e., fromwhich the putative crRNA was identified) using Bowtie 2(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).

The Sequence Alignment/Map (SAM) file generated by Bowtie 2 can beconverted into a Binary Alignment/Map (BAM) file using SAMTools(http://samtools.sourceforge.net/) for subsequent sequencing analysissteps.

Read coverage mapping to the CRISPR locus or loci can be calculated fromthe BAM file using BedTools (bedtools.readthedocs.org/en/latest/).

The BED file, generated in the previous step, can be loaded intoIntegrative Genomics Viewer (IGV; www.broadinstitute.org/igv/) tovisualize the sequencing read pileup. Read pile-ups can be used toidentify the 5′ and 3′ ends of the transcribed putative tracrRNAsequence.

The RNA-seq data can be used to validate that a putative tracrRNAelement sequence is actively transcribed in vivo. Confirmed hits fromthe comparison of the in silico and RNA-seq screens can be validated forfunctional ability of the identified tracrRNA sequence and its cognatecrRNA to support Cas9-mediated cleavage of a double-stranded DNA targetsequence using methods outline herein (e.g., Examples 2, 3, and 5).

Following the guidance of the present specification and Examples, theidentification of tracrRNA sequences related to Cas9 proteins can beaccomplished by one of ordinary skill in the art.

EXAMPLE 9 T7E1 Assay for Detection of Target Sequence Modifications inEukaryotic Cells

This Example illustrates the use of T7E1 assays to evaluate and comparethe percent cleavage in vivo of NASC/Cas9 protein complexes (e.g.,NASC-PC1/NASC-PC2/Cas9 protein complexes) relative to selecteddouble-stranded DNA target sequences.

A. Cell Transfections Using Cas Polynucleotide Components

NASC-PC1 and NASC-PC2 can be transfected into HEK293 cellsconstitutively expressing S. pyogenes Cas9 (HEK293-Cas9), using theNucleofector® 96-well Shuttle System (Lonza, Allendale, N.J.) and thefollowing protocol. NASC-PC1 and NASC-PC2 can be individually diluted toappropriate concentration (e.g., 120 pmol), mixed together, incubatedfor 2 minutes at 95° C., removed from the thermocycler, allowed toequilibrate to room temperature, and dispensed in a 5 μL final volume ina 96-well plate. Culture medium can be aspirated from HEK293-Cas9 cells,the cells can be washed once with calcium and magnesium-free PBS, andcan be trypsinized by the addition of TrypLE (Life Technologies, GrandIsland, N.Y.) followed by incubation at 37° C. for 3-5 minutes.Trypsinized cells can be gently pipetted up and down to form asingle-cell suspension and added to DMEM complete culture mediumcomposed of DMEM culture medium (Life Technologies, Grand Island, N.Y.)containing 10% Fetal Bovine Serum (FBS; Thermo Scientific, Wilmington,Del.) and supplemented with penicillin and streptomycin (LifeTechnologies, Grand Island, N.Y.).

The cells can be pelleted by centrifugation for 3 minutes at 200×g, theculture medium can be aspirated, and cells can be re-suspended in PBS.The cells can be counted using the Countess® II Automated Cell Counter(Life Technologies, Grand Island, N.Y.). 2.2×10⁷ cells can betransferred to a 1.5 ml microfuge tube and pelleted. The PBS can beaspirated and the cells can be re-suspended in Nucleofector™ SF (Lonza,Allendale, N.J.) solution to a density of 1×10⁷ cells/mL. 20 μL of thecell suspension can be added to individual wells containing 5 uL ofNASC-PC1/NASC-PC2 and the entire volume can be transferred to the wellsof a 96-well Nucleocuvette™ Plate (Lonza, Allendale, N.J.). The platecan be loaded onto the Nucleofector™ 96-well Shuttle™ (Lonza, Allendale,N.J.) and cells can be nucleofected using the 96-CM-130 Nucleofector™program (Lonza, Allendale, N.J.). Post-nucleofection, 70 μL DMEMcomplete culture medium can be added to each well, and 50 μL of the cellsuspension can be transferred to a collagen coated 96-well cell cultureplate containing 150 μL pre-warmed DMEM complete culture medium. Theplate can be transferred to a tissue culture incubator and maintained at37° C. in 5% CO₂ for 48 hours.

B. Double-Stranded DNA Target Sequence Generation for T7E1 Assay

gDNA can be isolated from HEK293-Cas9 cells 48 hours after transfectionwith NASC-PC1/NASC-PC2 using 50 μL QuickExtract DNA Extraction solution(Epicentre, Madison, Wis.) per well followed by incubation at 37° C. for10 minutes, 65° C. for 6 minutes and 95° C. for 3 minutes to stop thereaction. gDNA can be diluted with 150 μL water and samples can bestored at −80° C.

DNA for T7E1 can be generated by PCR amplification of double-strandedDNA target sequences (e.g., XRCC5_T1 and XRCC5_T3) from isolated gDNA.PCR reactions can be set up using 8 μL gDNA as template with KAPA HiFiHot Start polymerase and contain 0.5 U of polymerase, 1× reactionbuffer, 0.4 mM dNTPs and 300 nM forward and reverse primers directed toone of the double-stranded DNA target sequences (e.g., SEQ ID NO. 47/SEQID NO. 48 and SEQ ID NO. 49/SEQ ID NO. 50) in a total volume of 25 μL.The DNA target sequences can be amplified using the followingconditions: 95° C. for 5 minutes, 4 cycles of 20 seconds at 98° C., 20seconds at 70° C., minus 2° C./cycle, 30 seconds at 72° C., followed by30 cycles of 15 seconds at 98° C., 20 seconds at 62° C., 20 seconds at72° C., and a final extension at 72° C. for 1 minute.

C. T7E1 Assay

PCR-amplified double-stranded DNA target sequences for T7E1 assays canbe denatured at 95° C. for 10 minutes and then allowed to re-anneal bycooling to 25° C. at −0.5° C./second in a thermal cycler. There-annealed DNA can be incubated with 0.5 μL T7 Endonuclease I in 1×NEBuffer 2 buffer (New England Biolabs, Ipswich, Mass.) in a totalvolume of 15 μL for 25 minutes at 37° C. T7E1 reactions can be analyzedusing the Fragment Analyzer™ System (Advanced Analytical Technologies,Ames, Iowa) and the DNF-910 Double-stranded DNA Reagent Kit (AdvancedAnalytical Technologies, Ames, Iowa). The Fragment Analyzer™ Systemprovides the concentration of each cleavage fragment and of thedouble-stranded DNA target sequence that remains after cleavage.

Cleavage percentages of the double-stranded DNA target sequences can becalculated from the concentration of each cleavage fragment and thedouble-stranded DNA target sequence that remains after cleavage hastaken place, using the following formula:

$\begin{matrix}{{\% \; {cleavage}} = \left( {1 - \sqrt{\left( {1 - \frac{\left( {{{frag}\; 1} + {{frag}\; 2}} \right)}{\left( {{{frag}\; 1} + {{frag}\; 2} + {parent}} \right)}} \right)}} \right)} & {{EQUATION}\mspace{14mu} 1}\end{matrix}$

In Equation 1, frag1 and frag2 concentrations correspond to theconcentration of Cas9 cleavage fragments of the double-stranded DNAtarget sequence and parent corresponds to the double-stranded DNA targetsequence that remains after cleavage has taken place.

The T7E1 assay for detection of target sequence modifications ineukaryotic cells provides data to demonstrate that the NASCpolynucleotide compositions described herein facilitate Cas9-mediatedsite-specific in vivo cleavage of multiple double-stranded DNA targetsequences. sgRNA, crRNA, and/or crRNA/tracrRNA polynucleotides havingthe same DNA target binding sequence as the NASC polynucleotidecomposition can also be included in the assay to compare theCas-mediated site-specific cleavage percentages between the constructs.

Following the guidance of the present specification and Examples, theT7E1 assay described in this Example can be practiced by one of ordinaryskill in the art with other Cas proteins and their cognate NASCpolynucleotide compositions.

EXAMPLE 10 Probing for Sites Tolerant of Modification in Class 2 Type VCpf1 Guide RNA Backbones

This Example describes the generation and testing of variousmodifications of Class 2 Type V guide crRNAs and their suitability foruse in constructing NASC polynucleotide components. The method describedbelow is adapted from Briner, A., et al., Molecular Cell 56(2):333-339(2014). Not all of the following steps are required for screening normust the order of the steps be as presented.

In this Example, modifications can be introduced into the crRNAbackbone, and the modified crRNA tested with a cognate Cpf1 nuclease tofacilitate identification of regions or positions in the Cpf1-crRNAbackbone wherein linkages for NASC polynucleotide components can beengineered.

A crRNA from a Class 2 Type V CRISPR system (e.g., Acidaminococcus sp.Cpf1) can be selected for engineering. The crRNA sequence can bemodified in silico to introduce one or more base substitutions,deletions, or insertions into nucleic acid sequences in regions selectedfrom one or more of the following regions: nucleic acid sequences 5′ ofthe pseudo-knot, Cpf1-stem RNA sequence 1, the pseudo-knot loop (loopelement nucleic acid sequence), Cpf1-stem RNA sequence 1C, or the spacerelement.

The crRNA sequence can be modified in silico to introduce one or morebreak in the phosphodiester backbone in one or more regions selectedfrom the following: nucleic acid sequences 5′ of the pseudo-knot,Cpf1-stem RNA sequence 1, the pseudo-knot loop (loop element nucleicacid sequence), Cpf1-stem RNA sequence 1C, or the spacer element.

Base modification can also be used to introduce mismatches in thehydrogen base-pair interactions of any of the crRNA regions, orbase-pair mutation introducing an alternative hydrogen base-pairinteraction through substitution of two bases, wherein the alternativehydrogen base-pair interaction differs from the original hydrogenbase-pair interaction (e.g., the original hydrogen base-pair interactionis Watson-Crick base pairing and the substitution of the two bases forma reverse Hoogsteen base pairing). Substitution of bases can also beused to introduce hydrogen base-pair interaction within the crRNAbackbone (e.g., within the pseudo-knot loop sequence).

Regions of the crRNA can be independently engineered to introducesecondary structure elements into the crRNA backbone. Such secondarystructure elements include, but are not limited to, the following:stem-loop elements, stem elements, pseudo-knots, and ribozymes.Furthermore, the crRNA guide RNA backbone can be modified to deleteportions of the crRNA backbone either through deletion at the 5′ end, 3′end or internal to the crRNA. Alternative backbone structures can alsobe introduced.

In silico designed crRNA sequences can be provided to a commercialmanufacturer for synthesis.

Modified crRNAs can be evaluated for their ability to support cleavageof a double-stranded DNA target sequence mediated by the cognate Cpf1protein to the crRNA that gave rise to the modified crRNA. Amplificationof double-stranded DNA target sequences and the biochemical cleavageassay can be carried out in a manner similar to those described inExample 4 and Example 5, respectively. Modified crRNA that are capableof mediating cleavage of a DNA target sequence with their cognate Cpf1proteins can be validated for activity in cells using the methoddescribed in Example 6.

Following the guidance of the present specification and Examples, themodification of a Cpf1 crRNA (e.g., introduction or deletion of varioussequences, and/or introduction or deletion of secondary structuralmodifications) can be used to probe for locations for insertion orlinkages to facilitate making NASC polynucleotide compositions. ThisExample can be practiced by one of ordinary skill in the art with otherType V CRISPR Cpf1 proteins and other Type V CRISPR crRNA in view of theteachings of the present specification.

EXAMPLE 11 Probing for Sites Tolerant of Modification in Class 2 Type IICas9 Guide RNA Backbones

This Example describes the generation and testing of variousmodifications of Class 2 Type II guide RNA(s) and their suitability foruse in constructing NASC polynucleotide compositions.

In this Example, modifications can be introduced into the RNA backboneof Class 2 Type II CRISPR guide RNA(s) (e.g., dual-guide RNAs orsingle-guide RNAs) to identify locations for engineering or attachmentof various nucleic acid sequences. The method described below is adaptedfrom Briner, A., et al., Molecular Cell 56(2):333-339 (2014). Not all ofthe following steps are required for screening nor must the order of thesteps be as presented.

A Class 2 Type II CRISPR sgRNA, crRNA, tracrRNA, or crRNA and tracrRNA(collectively referred to a “Cas9 guide RNA”) can be selected forengineering.

The Cas9 guide RNA sequence can be modified in silico to introduce oneor more base substitutions, deletions, or insertions into regionsselected from one or more of the following: a nucleic acid targetbinding sequence, a lower stem nucleic acid sequence, a bulge nucleicacid sequence, an upper stem nucleic acid sequence, a first stem-loopelement nucleic acid sequence, a nexus nucleic acid sequence, a linkingnucleic acid sequence, and/or 3′ hairpins. The Cas9 guide RNA sequencecan be modified in silico to introduce one or more breaks in thephosphodiester backbone in one or more regions selected from thefollowing: a nucleic acid target binding sequence, a lower stem nucleicacid sequence, a bulge nucleic acid sequence, an upper stem nucleic acidsequence, a first stem-loop element nucleic acid sequence, a nexusnucleic acid sequence, a linking nucleic acid sequence, and 3′ hairpins.

Base modification can be used to introduce mismatches in the hydrogenbase-pair interactions of any of the Cas9 guide RNA regions. Base-pairmutation can be used to introduce an alternative hydrogen base-pairinteraction through substitution of two bases, wherein the alternativehydrogen base-pair interaction differs from the original hydrogenbase-pair interaction (e.g., the original hydrogen base-pair interactionis Watson-Crick base pairing and the substitution of the two bases forma reverse Hoogsteen base pairing). Substitution of bases can also beused to introduce hydrogen base-pair interaction within the Cas9 guideRNA backbone (e.g., within the bulge sequence).

Regions of the Cas9 guide RNA can be independently engineered tointroduce secondary structure elements into the Cas9 guide RNA backbone.Such secondary structure elements include, but are not limited to, thefollowing: stem-loop elements, stem elements, pseudo-knots, andribozymes. Furthermore, the Cas9 guide RNA backbone can be modified todelete portions of the Cas9 guide RNA backbone through deletion at the5′ end, 3′ end, and/or or internal to the Cas9 guide RNA. Alternativebackbone structures can also be introduced.

In silico designed Class 2 Type II CRISPR Cas9 guide RNA sequences canbe provided to a commercial manufacturer for synthesis.

Modified Class 2 Type II CRISPR Cas9 guide RNAs can be evaluated forability to support cleavage of a double-stranded DNA target sequencemediated by the cognate Cas9 protein to the Cas9 guide RNA that gaverise to the modified Cas9 guide RNA. Amplification of a double-strandedDNA target sequences and the biochemical cleavage assay can be carriedout in a manner similar to those described in Example 4 and Example 5,respectively. Modified Cas9 guide RNAs capable of mediating cleavage ofa DNA target sequence with their cognate Cas9 proteins can be validatedfor activity in cells using the method described in Example 6.

Following the guidance of the present specification and Examples, themodification of a Cas9 guide RNA(s) (e.g., introduction or deletion ofvarious sequences, and/or introduction or deletion of secondarystructural modifications) can be used to probe for locations forinsertion or linkages to facilitate making NASC polynucleotidecompositions. This Example can be practiced by one of ordinary skill inthe art with other Type II CRISPR Cas9 proteins and other Type II CRISPRCas9 guide RNA in view of the teachings of the present specification.

EXAMPLE 12 Screening of NASC Polynucleotide Compositions Comprising DNATarget Binding Sequences

This Example illustrates the use of NASC polynucleotide compositions ofthe present invention to modify DNA target sequences present in humangDNA and measure the level of cleavage activity at those sites.

Target sites (DNA target sequences) can be first selected from gDNA.Individual components of NASC polynucleotide compositions can bedesigned to target the selected sequences. Assays (e.g., as described inExample 5) can be performed to determine the level of DNA targetsequence cleavage.

Not all of the following steps are required for every screening nor mustthe order of the steps be as presented, and the screening can be coupledto other experiments, or form part of a larger experiment.

A. Selecting DNA Target Regions (DNA Target Sequences) from gDNA

PAM sequences (i.e., NGG, TTN, etc.) for a Cas protein (e.g., S.pyogenes Cas9 or Acidaminococcus sp. Cpf1) can be identified within theselected genomic region.

One or more Cas9 DNA target sequences (20 nucleotides in length) thatare 5′ adjacent to a PAM sequence can be identified and selected or oneor more Cpf1 DNA target sequences (20-24 nucleotide in length) that are3′ adjacent to a PAM sequence can be identified and selected.

Criteria for selection of nucleic acid target sequences can include, butare not limited to, the following: homology to other regions in thegenome, percent G-C content, melting temperature, presences ofhomopolymer within the spacer, distance between the two sequences, andother criteria known to one skilled in the art.

If a Type II CRISPR NASC polynucleotide composition is desired to beused, the DNA target binding sequence can be incorporated at the 5′ end.If a Type V CRISPR NASC polynucleotide composition is desired to beused, the DNA target binding sequence can be incorporated at the 3′ end.A commercial manufacturer typically synthesizes NASC polynucleotidecompositions based on provided sequences. Alternatively, the NASCpolynucleotide compositions can be produced as described in Example 2 byin vitro transcription.

NASC polynucleotide compositions as described herein can be used withcognate Class 2 Type II CRISPR nuclease (e.g., a Cas9 nuclease), a Class2 Type V CRISPR nuclease (e.g., a Cpf1 nuclease), or both a cognateClass 2 Type II CRISPR nuclease and a Class 2 Type V CRISPR nuclease toform NASC/Cas protein complexes.

B. Determination of Cleavage Percentages and Specificity

In vitro cleavage percentages and specificity (e.g., the amount ofoff-target binding) related to NASC polynucleotide compositions can bedetermined, for example, using the cleavage assays described in Example5 and can be compared as follows:

(1) If only a single pair of DNA target sequences can be identified orselected for a NASC, the cleavage percentage and specificity for each ofthe DNA target sequences can be determined. If so desired, cleavagepercentage and/or specificity can be altered in further experimentsusing methods including, but not limited to, modifying the NASC; orintroducing effector proteins/effector protein-binding sequences tomodify the NASC, a NASC polynucleotide component, or the Cas protein; orintroducing ligand/ligand binding moieties to modify the NASCpolynucleotide or the Cas protein.

(2) If multiple pairs of DNA target sequences can be identified orselected for a NASC, the percentage cleavage data and site-specificitydata obtained from the cleavage assays can be compared between differentDNAs comprising the target binding sequence to identify the DNA targetsequences having the desired cleavage percentage and specificity.Cleavage percentage data and specificity data provide criteria on whichto base choices for a variety of applications. For example, in somesituations the activity of the NASC polynucleotide composition may bethe most important factor. In other situations, the specificity of thecleavage site may be relatively more important than the cleavagepercentage. If so desired, cleavage percentage and/or specificity can bealtered in further experiments using methods including, but not limitedto, modifying the NASC; or introducing effector proteins/effectorprotein-binding sequences to modify the NASC, a NASC polynucleotidecomponent, or the Cas protein; or introducing ligand/ligand bindingmoieties to modify the NASC polynucleotide component or the Cas protein.

Alternatively, or in addition to the in vitro analysis, in cell cleavagepercentages and specificities associated with NASC polynucleotidecompositions can be obtained using, for example, the method described inExample 6, can be compared as follows:

(1) If only a single pair of DNA target sequences can be identified orselected for a NASC, the cleavage percentage and specificity for each ofthe DNA target sequences can be determined. If so desired, cleavagepercentage and/or specificity can be altered in further experimentsusing methods including, but not limited to, modifying the NASC; orintroducing effector proteins/effector protein-binding sequences tomodify the NASC, a NASC polynucleotide component, or the Cas protein; orintroducing ligand/ligand binding moieties to modify the NASCpolynucleotide component or the Cas protein.

(2) If multiple pairs of DNA target sequences can be identified orselected for a NASC, the percentage cleavage data and site-specificitydata obtained from the cleavage assays can be compared between differentDNAs comprising the target binding sequence to identify the DNA targetsequences having the desired cleavage percentage and specificity.Cleavage percentage data and specificity data provide criteria on whichto base choices for a variety of applications. For example, in somesituations the activity of the NASC polynucleotide composition may bethe most important factor. In other situations, the specificity of thecleavage site may be relatively more important than the cleavagepercentage. If so desired, cleavage percentage and/or specificity can bealtered in further experiments using methods including, but not limitedto, modifying the NASC; or introducing effector proteins/effectorprotein-binding sequences to modify the NASC, a NASC polynucleotidecomponent, or the Cas protein; or introducing ligand/ligand bindingmoieties to modify the NASC polynucleotide component or the Cas protein.

Following the guidance of the present specification and Examples, thescreening described in this Example can be practiced by one of ordinaryskill in the art with other NASC polynucleotide compositions for usewith cognate Class 2 Type II CRISPR Cas9 proteins, cognate Class 2 TypeV CRISPR Cpf1 proteins, or both cognate Class 2 Type II CRISPR Cas9proteins and cognate Class 2 Type V CRISPR Cpf1 proteins.

EXAMPLE 13 Engineering of Ribonucleoprotein Closed-Cage ComplexesComprising NASC Polynucleotide Compositions

This Example illustrates the use of NASC polynucleotide compositions ofthe present invention for formation of NASC-CC closed-cage complexes forpackaging of small molecules.

A NASC-CC can be engineered, for example, using a first NASCpolynucleotide composition and a second NASC polynucleotide composition,each having the general structure shown in FIG. 6H (FIG. 6H, I,NASC-PC1; FIG. 6H, II, NASC-PC2; and FIG. 6H, III, NASC-PC-3). The firstNASC polynucleotide composition and the second NASC polynucleotidecomposition can be used in combination with three double-stranded DNAsequences. Each double-stranded DNA sequence (“a double-stranded DNAbrace sequence”) can comprise two unique DNA target sequences, whereinthe first DNA target sequence is complementary to a first nucleic acidbinding sequence of the first NASC polynucleotide composition, and thesecond DNA target sequence is complementary to a second nucleic acidbinding sequence of the second NASC polynucleotide composition.

NASC-CC and associated Cas proteins can be used to create closed-cagecomplexes suitable for the packaging of molecules. The size of the cagecan be varied by changing the design of the NASC-CC components or bybinding different length DNA target sequences.

A. Design of NASC-CC Components

A first NASC polynucleotide composition (referred to in this Example asa “NASC-triplex1”) can be engineered comprising a NASC-PC1, a NASC-PC2,and a NASC-PC3, which are similar in structure to those depicted in FIG.6A (referred to in this Example as a “NASC-PC1-triplex1,” a“NASC-PC2-triplex1,” and a “NASC-PC3-triplex1”). A first 20-nucleotideDNA target sequence can be added to the 5′ end (see, e.g., FIG. 6A,610-611) of each of NASC-PC1-triplex1, NASC-PC2-triplex1, andNASC-PC3-triplex1. The DNA target sequence typically will be selected tohave no or limited homology to native DNA sequences in an organism intowhich the NASC-CC are to be introduced (e.g., human gDNA or plant gDNA).

A second NASC polynucleotide composition (referred to in this Example as“NASC-triplex2”) can be engineered comprising a NASC-PC1-triplex2, aNASC-PC2-triplex2, and a NASC-PC3-triplex2, which are similar instructure to those depicted in FIG. 6A. A second 20-nucleotide DNAtarget sequence can be added to the 5′ end (see, e.g., FIG. 6A, 610-611)of each of NASC-PC1-triplex2, NASC-PC2-triplex2, and NASC-PC3-triplex2.The DNA target sequence typically will be selected to have no or limitedhomology to native DNA sequences in an organism into which the NASC-CCare to be introduced (e.g., human gDNA or plant gDNA). Furthermore, the20-nucleotide DNA target sequences should be distinct from (i.e., notcomplementary to) the DNA target sequences engineered in theNASC-triplex1.

Illustrative components of NASC-triplex1 and NASC-triplex2 are presentedin Table 20. In the table, the “Target sequence” column indicates the 20bp DNA target sequence that is complementary to the nucleic acid targetbinding sequence in the corresponding NASC polynucleotide component.

TABLE 20 NASC-triplex 1 and NASC-triplex2 Components NASC- NASC TargetSEQ triplex component sequence Sequence* ID NO. NASC- NASC- 1AUCUUGUUGACACGAGGAAUGU SEQ triplex1 PC1- UUUAGUCCCUAAUUAAAUUUCU ID NO.triplex1 UGAAAUUGGUAUAUAAGGAGGG 57 ACUACAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCC UAAAACCGCUUUUAAAAUUCAA AUAAAUUUUGCUUU NASC- NASC-1 AUCUUGUUGACACGAGGAAUGU SEQ triplex1 PC2- UGUAGUCCCUCCUUAUAUACCA ID NO.triplex1 AGAAAAAGAAAUUUAAAACUGA 58 ACUCCAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCC UAAAACCGCUUUUAAAAUUCAA AUAAAUUUUGCUUU NASC- NASC-1 AUCUUGUUGACACGAGGAAUGU SEQ triplex1 PC3- UGGAGUUCAGUUUUAAAUUUCU ID NO.triplex1 UGAAAAAGAAAUUUAAUUAGGG 59 ACUAAAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCC UAAAACCGCUUUUAAAAUUCAA AUAAAUUUUGCUUU NASC- NASC-2 CGAUAUAAUACAGCAAGGUGGU SEQ triplex2 PC1- UUUAGACCCCUCUUCCAUUUCGCID NO. triplex2 GAAAGCGUUUUGAGAGAGUGAA 60 CUACAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCU AAAACCGCUUUUAAAAUUCAAA UAAAUUUUGCUUU NASC- NASC-2 CGAUAUAAUACAGCAAGGUGGU SEQ triplex2 PC2- UGUAGUUCACUCUCUCAAAACG ID NO.triplex2 CGAAAAAGAAAUUUAAUAAGGA 61 ACUACAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCC UAAAACCGCUUUUAAAAUUCAA AUAAAUUUUGCUUU NASC- NASC-2 CGAUAUAAUACAGCAAGGUGGU SEQ triplex2 PC3- UGUAGUUCCUUAUUAAAUUUCU ID NO.triplex2 UGAAAGCGAAAUGGAAGAGGGG 62 UCUAAAACAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCC UAAAACCGCUUUUAAAAUUCAA AUAAAUUUUGCUUU*NASC-triplex hybridizing regions are underlined

A double-stranded DNA brace sequence can be engineered to incorporate,in the 5′ to 3′ direction, a 20 nucleotide random sequence at the 5′end, target sequence 1, the C. jejuni PAM sequence 5′-NNNACA-3′ (where“N” is any nucleotide), 50 nucleotides of random sequence, the reversecompliment of the C. jejuni PAM sequence, the reverse compliment oftarget sequence 2, and a randomize 20-nucleotide sequence at the 3′ end.The double-stranded DNA brace sequence will be targetable by C. jejunidCas9 proteins when bound to both the NASC-PC1-triplex1 and theNASC-PC1-TRP2 and will bring the two NASCs within proximity of oneanother. The sequence of the double-stranded DNA brace sequence can beprovided to a commercial manufacturer for synthesis of thedouble-stranded DNA. Alternatively, the sequence of the double-strandedDNA brace sequence can be constructed using single-stranded DNAoligonucleotides, similar to the construction of double-stranded DNAtemplate presented in Example 2.

An illustrative sequence for a double-stranded DNA brace sequence isshown in Table 21.

TABLE 21 Double-stranded DNA Brace Sequence SEQ ID Sequence* NO.CGTCGCTATGATTTGCCTATATCTTGTTGACACGAGGAAT SEQ IDGTAAACAACGAGTTCCGCTATTGGGATGGAGTTTAACTGT NO.CGCAACTCTCATCGCAATGTCAGTCACCTTGCTGTATTAT 63 ATCGCGCATGATAAAGTACGCCAT*Target and PAM sequences are bolded

B. Engineering and Production of C. jejuni dCas9 Protein

A C. jejuni (e.g., C. jejuni NCTC 1168; SEQ ID NO. 103) Cas9 amino acidsequence can be mutated from an aspartic acid at amino acid position 8to an alanine (D8A) and a histidine at position 559 to alanine(D8A/H559A) to generate a nuclease-inactive form of the C. jejuni Cas9protein (C. jejuni dCas9 protein; SEQ ID NO. 56). C. jejuni dCas9protein will remain capable of binding to a NASC-triplex1. Three C.jejuni dCas9 proteins are capable of binding to the NASC-triplex1 anddirecting the NASC-triplex1 to bind the target sequences complementaryto the nucleic acid target binding sequences therein. The C. jejunidCas9 protein can be C-terminally tagged with two nuclear localizationsequences (NLS) and can be recombinantly expressed in E. coli, andpurified using chromatographic methods.

C. Formation of NASC-CCs

NASC-triplex1 can be formed by mixing NASC-PC1-triplex1,NASC-PC2-triplex1, and NASC-PC3-triplex1 (Table 20) in equal molarconcentration, incubating for 2 minutes at 95° C., annealing by coolingto 25° C. at −0.5° C./second in a thermal cycler, and then allowing themixture to equilibrate to room temperature. NASC-triplex2 can be formedby mixing NASC-PC1-triplex2, NASC-PC2-triplex2, and NASC-PC3-triplex2(Table 20) in equal molar concentration, incubating for 2 minutes at 95°C., annealing by cooling to 25° C. at −0.5° C./second in a thermalcycler, and then allowing the mixture to equilibrate to roomtemperature.

Ribonucleoprotein closed-cage complexes can be formed by mixingNASC-triplex1 in the presence of an excess concentration of the C.jejuni dCas9 protein in a binding buffer (20 mM HEPES, 100 mM KCl, 5 mMMgCl₂, and 5% glycerol at pH 7.4) and incubating at 37° C. for 20minutes. The double-stranded DNA brace sequence can be added at alimiting concentration to the mixture comprising the NASC-triplex1/dCas9protein complex, and incubated for 37° C. for 20 minutes. NASC-triplex2can be added to the mixture of NASC-triplex1/dCas9protein/double-stranded DNA brace sequences at an equivalentconcentration of NASC-triplex1. The mixture can be incubated for 1 hourat 37° C. NASC-triplex1/dCas9 protein/double-stranded DNA bracesequences/NASC-triplex2/dCas9 protein closed-cage complexes can befrozen at −80° C. for long-term storage.

FIG. 6L illustrates an example of an underlying nucleic acid scaffoldstructure (NASC-CC), with Cas proteins omitted for clarity.NASC-triplex1 and NASC-triplex2 are the structures in this figure thatcorrespond to the structure shown in FIG. 6G. The dashed lines in thisfigure give an indication of the kinds of connections created by thedouble-stranded DNA brace sequences between NASC-triplex1 andNASC-triplex2. FIG. 6M illustrates the NASC-CC in complex with the dCas9protein proteins. The dCas9 protein proteins are represented in thisfigure by the grey circles.

Following the guidance of the present specification and Example, theformulation of other NASC-CC ribonucleoprotein closed-cage complexes(e.g., comprising various combinations of the NASC compositionsdescribed herein) can be practiced by one of ordinary skill in the artwith other NASC compositions and cognate Cas proteins.

EXAMPLE 14 Structural Analysis of NASC Ribonucleoprotein Closed-CageComplexes

The following Example describes characterization of NASC-CC/dCas proteinclosed-cage complexes to verify proper assembly and assess the size andvolume of assembled NASC-CC/dCas protein complexes. The method describedbelow is adapted from Andersen, F., et al., Nucleic Acids Research36(4):1113-1119 (2008) and Lapinaite, A., et al., Nature502(7472):519-523 (2013). Not all of the following steps are requiredfor screening nor must the order of the steps be as presented.

A. Electrophoretic Mobility Shift Assay of NASC-CC/dCas ProteinComplexes

NASC-CC/dCas protein complexes can be formulated as described in Example13, modified so that a radiolabeled double-stranded DNA brace sequencecan be used. The double-stranded DNA brace sequence can be radiolabeledby preparing the following reaction mixture: double-stranded DNA bracesequences in the presence of T4 polynucleotide kinase (New EnglandBiolabs, Ipswich, Mass.), γ-(³²P) ATP (Promega, Madison, Wis.), and 1×T4 polynucleotide kinase reaction buffer. The reaction mixture can beincubated and then heat inactivated at 65° C. for 20 minutes.Radiolabeled DNA can be purified using an Illustra MicroSpin G-25 column(GE Healthcare, Pittsburgh, Pa.).

Alternatively, one or more of the NASC-CC components can be radiolabeledin a similar manner.

Radiolabeled NASC-CC/dCas9 protein complexes can be aliquoted into a 10μL volume, and resolved at 4° C. by electrophoresis in a 8% nativepolyacrylamide gel containing 1× Tris/Borate/EDTA buffer (90 mM Tris, 90mM boric acid, 2 mM EDTA at pH 8.3) and 5 mM MgCl₂. The gel can besubsequently dried and imaged using the PMI™ system (Bio-RadLaboratories, Hercules, Calif.). Individual polynucleotide components ofthe NASC-CC (e.g., NASC-PC1-triplex1, NASC-PC2-triplex1,NASC-PC3-triplex1, NASC-PC1-triplex2, NASC-PC2-triplex2,NASC-PC3-triplex2, double-stranded DNA brace sequences, and/orindividual components complexed with dCas9 protein) can be used ascontrols for comparison to identify the electrophoretic mobility shiftof the completely formed NASC-CC/dCas9 protein complexes.

B. Small Angle X-ray Scattering of NASC-CC/dCas9 Protein Complexes

NASC-CC/dCas9 protein complexes, described in Example 13, can bedialyzed at 4° C. in a buffer of 20 mM HEPES, 100 mM KCl, 5 mM MgCl₂,and 5% glycerol at pH 7.4. The dialyzed preparation of NASC-CC/dCas9protein complexes can be dispensed into the wells of a 96-well plateusing a concentration series from 1 mg/mL to 5 mg/mL in a final volumeof 40 μL.

Small angle X-ray scattering (SAXS) measurements can be collected at aservice provider, such as The Advanced Light Source (Berkeley, Calif.),using a Structurally Integrated BiologY for Life Sciences (SIBYLS)beamline with a Mar165CCD detector. Data can be collected in multipleframes with exposure time ranges of 0.5 second to 10 seconds anddetector distances of 1.5 meters to 5 meters. Optimal collectionconditions can be evaluated for minimal radiation damage to sample aswell as optimal signal to noise ratios. Similarly, beamlinekiloelectron-volts (keV) energy can be tuned from a range of 7 keV to 15keV. Buffer-only control can be used as background and subtracted frommeasurements.

Data processing and analysis can be performed using standard beamlinesoftware and PRIMUS (Konarev, P., et al., Journal of AppliedCrystallography 36:1277-1282 (2003)). Data modeling can be performedusing SAXS analysis programs, such as an open source software suite(e.g., ATSAS 2.7.2, Petoukhov, M., et al., Journal of AppliedCrystallography 45:342-350 (2012)). Atomic coordinates of Cas9 proteinand single-guide RNA in different nucleotide bound states (e.g., sgRNAonly, sgRNA plus target strand, sgRNA plus target and non-targetstrand), as well as structures (e.g., nucleases, proteins,double-stranded DNA and RNA) are available from the Protein Database(PDB, www.rcsb.org/pdb/home/home.do) or Electron Microscopy Data Bank(EMDB, www.ebi.ac.uk/pdbe/emdb/). These atomic coordinates can be usedto calculate the internal volume, pore size, and closed-cage sizes ofthe NASC-CC/dCas9 protein complexes by modeling, combined with SAXSdata.

NASC-CC/dCas9 protein complexes can be modified to increase or decreasethe internal volume, pore size, or closed-cage sizes as needed for thepackaging and delivery of biomolecules, proteins, or other payloads.Such modifications can include, but are not limited to, lengthening orshortening of the first stem element nucleic acid sequence (FIG. 6A,608-609; FIG. 6H, 623-624/658-657, 626-625/627-628, 652-651/655-646),and/or the double-stranded DNA brace sequence (Table 21).

Following the guidance of the present specification and Examples,analysis of the structural features of NASC-CC/Cas protein complexes,including internal volumes, pore sizes, and closed-cage sizes, can bepracticed by one of ordinary skill in the art.

As is apparent to one of skill in the art, various modification andvariations of the above embodiments can be made without departing fromthe spirit and scope of this invention. Such modifications andvariations are within the scope of this invention.

1-20. (canceled)
 21. A complex of three engineered nucleic acids forminga scaffold, comprising: a first engineered nucleic acid, having a 5′ endand a 3′ end, comprising, a first Class 2 Type II CRISPR element 1,comprising, a first Class 2 Type II CRISPR protein binding sequence, anda repeat nucleic acid sequence 1 located 5′ of the first Class 2 Type IICRISPR protein binding sequence, the repeat nucleic acid sequence 1comprising, in a 3′ to 5′ direction, a linker element nucleic acidsequence 1-1, a repeat nucleic acid sequence 1a, a linker elementnucleic acid sequence 1-2, a repeat nucleic acid sequence 1b, and, alinker element nucleic acid sequence 1-3, and, a second Class 2 Type IICRISPR element 1, comprising a nucleic acid sequence 1 located 5′ of therepeat nucleic acid sequence 1, wherein the nucleic acid sequence 1comprises a spacer nucleic acid sequence 1; a second engineered nucleicacid, having a 5′ end and a 3′ end, comprising, a first Class 2 Type IICRISPR element 2, comprising a second Class 2 Type II CRISPR proteinbinding sequence, and a repeat nucleic acid sequence 2, located 5′ ofthe second Class 2 Type II CRISPR protein binding sequence, the repeatnucleic acid sequence 2 comprising, in a 5′ to 3′ direction, a linkerelement nucleic acid sequence 2-3, a repeat nucleic acid sequence 1bC, alinker element nucleic acid sequence 2-4, a repeat nucleic acid sequence2a, and a linker element nucleic acid sequence 2-5, and, a second Class2 Type 2 CRISPR element 2, comprising a nucleic acid sequence 2 located5′ of the repeat nucleic acid sequence 2, wherein the nucleic acidsequence 2 comprises a spacer nucleic acid sequence 2; and a thirdengineered nucleic acid, having a 5′ end and a 3′ end, comprising, afirst Class 2 Type II CRISPR element 3, comprising, a third Class 2 TypeII CRISPR protein binding sequence, and a repeat nucleic acid sequence 3located 5′ of the third nucleic acid binding Class 2 Type II CRISPRprotein binding sequence, the repeat nucleic acid sequence 3 comprising,in a 3′ to 5′ direction, a linker element nucleic acid sequence 3-1, arepeat nucleic acid sequence 2aC, a linker element nucleic acid sequence3-2, a repeat nucleic acid sequence 1aC, and a linker element nucleicacid sequence 3-3, and, a second Class 2 Type II CRISPR element 3,comprising a nucleic acid sequence 3 located 5′ of the repeat nucleicacid sequence 3, wherein the nucleic acid sequence 3 comprises a spacernucleic acid sequence 3; wherein the repeat nucleic acid sequence la isconnected by base-pair hydrogen bonding to the repeat nucleic acidsequence 1aC, the repeat nucleic acid sequence 1b is connected bybase-pair hydrogen bonding to the repeat nucleic acid sequence 1bC, andthe repeat nucleic acid sequence 2a is connected by base-pair hydrogenbonding to the repeat nucleic acid sequence 2aC.
 22. The complex ofclaim 21, wherein a region of hydrogen-bond interactions between therepeat nucleic acid sequence la and the repeat nucleic acid sequence 1aCcomprises a first double-stranded nucleic acid binding protein bindingsite, a region of hydrogen-bond interactions between the repeat nucleicacid sequence 1b and the repeat nucleic acid sequence 1bC comprises asecond double-stranded nucleic acid binding protein binding site, aregion of hydrogen-bond interactions between the repeat nucleic acidsequence 2a and the repeat nucleic acid sequence 2aC comprises a thirddouble-stranded nucleic acid binding protein binding site, or acombination thereof.
 23. The complex of claim 22, wherein the firstdouble-stranded nucleic acid binding protein site, the seconddouble-stranded nucleic acid binding protein site, the thirddouble-stranded nucleic acid binding protein site, or a combinationthereof comprises a Csy4 protein binding protein site.
 24. The complexof claim 21, wherein the repeat nucleic acid sequence la furthercomprises, in a 3′ to 5′ direction, a repeat nucleic acid sequence 1a1,a bulge nucleic acid sequence 1a1, and a repeat nucleic acid sequence1a2; the repeat nucleic acid sequence 1aC further comprises, in a 3′ to5′ direction, a repeat nucleic acid sequence 1a2C, a bulge nucleic acidsequence 1a2, and a repeat nucleic acid sequence 1a1C; the repeatnucleic acid sequence 1b further comprises, in a 3′ to 5′ direction, arepeat nucleic acid sequence 1b1, a bulge nucleic acid sequence 1b1, anda repeat nucleic acid sequence 1b2; the repeat nucleic acid sequence 1bCfurther comprises, in a 3′ to 5′ direction, a repeat nucleic acidsequence 1b2C, a bulge nucleic acid sequence 1b2, and a repeat nucleicacid sequence 1b1C; the repeat nucleic acid sequence 2a furthercomprises, in a 3′ to 5′ direction, a repeat nucleic acid sequence 2a1,a bulge nucleic acid sequence 2a1, and a repeat nucleic acid sequence2a2; and the repeat nucleic acid sequence 2aC further comprises, in a 3′to 5′ direction, a repeat nucleic acid sequence 2a2C, a bulge nucleicacid sequence 2a2, and a repeat nucleic acid sequence 2a1C; wherein therepeat nucleic acid sequence 1a1 is connected by base-pair hydrogenbonding to the repeat nucleic acid sequence 1a1C, the repeat nucleicacid sequence 1a2 is connected by base-pair hydrogen bonding to therepeat nucleic acid sequence 1a2C, the repeat nucleic acid sequence 1b 1is connected by base-pair hydrogen bonding to the repeat nucleic acidsequence 1b1C, the repeat nucleic acid sequence 1b2 is connected bybase-pair hydrogen bonding to the repeat nucleic acid sequence 1b2C, therepeat nucleic acid sequence 2a1 is connected by base-pair hydrogenbonding to the repeat nucleic acid sequence 2a1C, and the repeat nucleicacid sequence 2a2 is connected by base-pair hydrogen bonding to therepeat nucleic acid sequence 2a2C.
 25. The complex of claim 24, whereina region of hydrogen-bond interactions between the repeat nucleic acidsequence 1a2 and the repeat nucleic acid sequence 1a2C comprises a firstdouble-stranded nucleic acid binding protein binding site, a region ofhydrogen-bond interactions between the repeat nucleic acid sequence 1b 1and the repeat nucleic acid sequence 1b1C comprises a seconddouble-stranded nucleic acid binding protein binding site, a region ofhydrogen-bond interactions between the repeat nucleic acid sequence 2a1and the repeat nucleic acid sequence 2a1C comprises a thirddouble-stranded nucleic acid binding protein binding site, or acombination thereof.
 26. The complex of claim 21, wherein the firstengineered nucleic acid comprises RNA, DNA, or a combination thereof;the second engineered nucleic acid comprises RNA, DNA, or a combinationthereof; and the third engineered nucleic acid comprises RNA, DNA, or acombination thereof.
 27. The complex of claim 21, wherein a first Class2 Type II CRISPR-Cas9 protein is capable of binding the first Class 2Type II CRISPR protein binding sequence and a region of hydrogen-bondinteractions between the repeat nucleic acid sequence 1a and the repeatnucleic acid sequence 1aC, a second Class 2 Type II CRISPR-Cas9 proteinis capable of binding the second Class 2 Type II CRISPR protein bindingsequence and a region of hydrogen-bond interactions between the repeatnucleic acid sequence 1b 1 and the repeat nucleic acid sequence 1b1C,and a third Class 2 Type II CRISPR-Cas9 protein is capable of bindingthe third Class 2 Type II CRISPR protein binding sequence a region ofhydrogen-bond interactions between the repeat nucleic acid sequence 2aand the repeat nucleic acid sequence 2aC.
 28. The complex of claim 27,wherein the first Class 2 Type II CRISPR-Cas9 protein, the second Class2 Type II CRISPR-Cas9 protein, and the third Class 2 Type II CRISPR-Cas9protein, or a combination thereof, comprises a Campylobacter jejuni Cas9protein.
 29. The complex of claim 25, wherein a first Class 2 Type IICRISPR-Cas9 protein is capable of binding (i) the first Class 2 Type IICRISPR protein binding sequence, (ii) a region of hydrogen-bondinteractions between the repeat nucleic acid sequence 1a1 and the repeatnucleic acid sequence 1a1C, (iii) a bulge region formed by the bulgenucleic acid sequence 1a1 and the bulge nucleic acid sequence 1a2, and(iv) a region of hydrogen-bond interactions between the repeat nucleicacid sequence 1a2 and the repeat nucleic acid sequence 1a2C, a secondClass 2 Type II CRISPR-Cas9 protein is capable of binding (i) the secondClass 2 Type II CRISPR protein binding sequence, (ii) a region ofhydrogen-bond interactions between the repeat nucleic acid sequence 1b2and the repeat nucleic acid sequence 1b2C, (iii) a bulge region formedby the bulge nucleic acid sequence 1b1 and the bulge nucleic acidsequence 1b2, and (iv) a region of hydrogen-bond interactions betweenthe repeat nucleic acid sequence 1b1 and the repeat nucleic acidsequence 1b1C, and a third Class 2 Type II CRISPR-Cas9 protein iscapable of binding (i) the third Class 2 Type II CRISPR protein bindingsequence, (ii) a region of hydrogen-bond interactions between the repeatnucleic acid sequence 2a2 and the repeat nucleic acid sequence 2a2C,(iii) a bulge region formed by the bulge nucleic acid sequence 2a1 andthe bulge nucleic acid sequence 2a2, and (iv) a region of hydrogen-bondinteractions between the repeat nucleic acid sequence 2a1 and the repeatnucleic acid sequence 2a1C.
 30. The complex of claim 29, wherein thefirst Class 2 Type II CRISPR-Cas9 protein, the second Class 2 Type IICRISPR-Cas9 protein, and the third Class 2 Type II CRISPR-Cas9 proteinare each selected from the group consisting of a Streptococcus pyogenesCas9 protein, a Streptococcus thermophilus Cas9 protein, and aStaphylococcus aureus Cas9 protein.
 31. The complex of claim 21, whereinthe spacer nucleic acid sequence 1 is complementary to a target nucleicacid sequence 1, the spacer nucleic acid sequence 2 is complementary toa target nucleic acid sequence 2, and the spacer nucleic acid sequence 3is complementary to a target nucleic acid sequence
 3. 32. The complex ofclaim 31, wherein the target nucleic acid sequence 1, the target nucleicacid sequence 2, and the target nucleic acid sequence 3 each comprisesdouble-stranded DNA.
 33. A nucleic acid/protein composition, comprising:the complex of claim 21; and a first Class 2 Type II CRISPR-Cas9protein, a second Class 2 Type II CRISPR-Cas9 protein, and a third TypeII CRISPR-Cas9 protein.
 34. The nucleic acid/protein composition ofclaim 33, wherein the first Class 2 Type II CRISPR-Cas9 protein, thesecond Class 2 Type II CRISPR-Cas9 protein, and the third Class 2 TypeII CRISPR-Cas9 protein are each selected from the group consisting of aStreptococcus pyogenes Cas9 protein, a Streptococcus thermophilus Cas9protein, a Staphylococcus aureus Cas9 protein, and a Campylobacterjejuni Cas9 protein.
 35. The nucleic acid/protein composition of claim33, wherein at least two of the first Class 2 Type II CRISPR-Cas9protein, the second Class 2 Type II CRISPR-Cas9 protein, and the thirdClass 2 Type II CRISPR-Cas9 protein are different Class 2 Type IICRISPR-Cas9 proteins, and each Class 2 Type II CRISPR-Cas9 protein isselected from the group consisting of a Streptococcus pyogenes Cas9protein, a Streptococcus thermophilus Cas9 protein, a Staphylococcusaureus Cas9 protein, and a Campylobacter jejuni Cas9 protein.
 36. Thenucleic acid/protein composition of claim 33, wherein at least one ofthe first Class 2 Type II CRISPR-Cas9 protein, the second Class 2 TypeII CRISPR-Cas9 protein, or the third Class 2 Type II CRISPR-Cas9 proteincomprises a catalytically inactive Cas9 protein.
 37. The nucleicacid/protein composition of claim 36, further comprising a donorpolynucleotide.
 38. The nucleic acid/protein composition of claim 33,wherein the complex comprises RNA, DNA, or RNA and DNA.
 39. A kit,comprising: the complex of claim 21; and a buffer.
 40. The kit of claim39, further comprising one or more Class 2 Type II CRISPR-Cas9 proteinsor one or more nucleic acid sequences encoding one or more Class 2 TypeII CRISPR-Cas9 proteins.